Antibody against periostin, and a pharmaceutical composition comprising it for preventing or treating a disease in which periostin is involved

ABSTRACT

The present invention provides an antibody against a periostin isoform having anti-cell adhesive activity, especially an anti-periostin antibody having the ability to neutralize anti-cell adhesive properties, as well as a prophylactic or therapeutic agent for periostin-related diseases comprising the antibody. The present invention also provides methods for detecting and quantifying the periostin isoform in a sample by using the antibody, as well as a method for diagnosing periostin-related diseases comprising measuring the amount of the periostin isoform by the detection or quantification method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International PatentApplication No. PCT/JP2006/326280, filed Dec. 28, 2006, which claimspriority to Japan 380009/2005, filed Dec. 28, 2005, Japan 169494/2007,filed Jun. 27, 2007, JP 33827/2008, Feb. 14, 2008, the disclosures ofeach of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

(i) Field of the Invention

The present invention relates to antibodies against periostin isoformshaving anti-cell adhesive properties, especially anti-periostinantibodies having the ability to neutralize anti-cell adhesiveproperties. More specifically, it relates to anti-periostin antibodiesspecifically recognizing a site responsible for anti-cell adhesion ofperiostin having anti-cell adhesive properties specifically expressed ininterstitial tissue during tissue restructuring such as cardiachypertrophy, which are useful for prevention or treatment ofperiostin-related diseases, such as heart failure, or are useful fordiagnosis of these diseases.

The present invention also relates to a pharmaceutical composition forcancer treatment, which comprises an antibody against a peptide encodedby the Exon-17 region of periostin. More specifically, the presentinvention relates to a pharmaceutical composition for cancer treatment,which comprises an anti-periostin antibody recognizing a splice variantof periostin having anti-cell adhesive properties specifically expressedin interstitial tissue during tissue restructuring such as cancertissues, or a method and reagent for cancer diagnosis using the aboveanti-periostin antibody.

(ii) Description of the Related Art

Chronic heart failure is a condition in which the heart cannot pumpenough blood to various organs due to decreased myocardialcontractility. Conventionally, it has been treated with cardiotonicdrugs that increase myocardial contractility such as digitalis drugs.However, these drugs have been shown to impair vital prognosis duringlong-term administration, due to excessive consumption of myocardialenergy. Thus, recently prevailing therapies are those using diuretics,β-blockers or angiotensin inhibitors that reduce excessive workload onthe heart by the sympathetic nervous system orrenin-angiotensin-aldosterone system activated in heart failurecondition. However, patients with heart failure have limited activitiesin their daily life and cannot maintain their quality of life becausethey are prohibited from hard exercise or the like. Moreover, the vitalprognosis of patients with heart failure cannot be fully ensured. It istherefore desirable to develop a new drug effective for treating heartfailure, which enables an improvement in the quality of life and animprovement in long-term vital prognosis.

In recent years, the healing rate of cancer has been rising steadilywith advances in cancer therapy. In particular, the improved successrate of primary carcinoma removal by surgical operation, radiationtherapy or chemotherapy contributes to the advance of cancer therapy.However, on a worldwide basis, the cancer mortality rate continues toincrease for reasons such as aging population, and cancer remains theprimary cause of death. This is because not a few patients will die ofcancer metastasis even when primary carcinoma removal is completelyachieved, and there is a limit to surgical operation, radiation therapyor chemotherapy for completely blocking cancer metastasis, so that thedistant metastasis of cancer is still directly or indirectly related tothe cause of cancer death. Cancer metastasis is mediated by processessuch as invasion of cancer cells released from their primary tumor intoblood vessels or lymph vessels, selective migration of cancer cells tometastatic organs, invasion of cancer cells from blood vessels intometastatic organs, growth of cancer cells supported by themicroenvironment where metastasis occurred, and angiogenesis-associatedgrowth of tumors whose diameter exceeds several millimeters (Folkman J.Semin. Cancer Biol, 3, 65-71, (1992), Hanahan D. et al. Cell, 86,353-364 (1996)). Among these complex processes for metastasisestablishment, invasion and metastasis induced by the enhanced motilityof cancer cells are very important stages (Liotta L A. et al. Cell, 64,327-336 (1991)). Until now, it has been reported that highly metastaticcancer cells produce an autocrine motility factor by themselves toenhance their own motion (Liotta L A. et al. Proc. Natl. Acad. Sci., 83,3302-3306 (1986)). Inhibitory substances against this malignant factorare expected as metastasis inhibitors, but no specific inhibitor hasbeen found at present.

On the other hand, periostin is an extracellular matrix protein andconsists of a polypeptide having a molecular weight of about 90000. Eachpolypeptide chain has a signal sequence, a cysteine-rich domain, afourfold repeated domain, and a C-terminal domain.

Periostin was first called osteoblast-specific factor-2 (OSF-2) and wasisolated and identified as a gene specifically expressed in the mouseosteoblast cell line MC3T3-E1 (JPA No. HEI-5-268982, Takeshita S. etal., Biochem J (1993) 294, 271-8), and later came to be known asperiostin and was reported to have adhesion-promoting activity inosteoblast cells (Horiuchi K. et J. Bone Miner. Res. (1999) 14,1239-49).

In early studies, periostin was thought to be an extracellular matrixspecifically expressed in bone tissue. However, it is currently known tobe expressed not only in bone tissue but also very highly at the onsetof heart failure (Katsuragi N. et al., Circulation (2004) 110, 1806-13,Wang D. et al., Hypertension (2003) 42, 88-95), aneurysms (Peters D G.et al., Stroke (2001) 32, 1036-42), highly metastatic cancers (Shao R.et al., Mol Cell Biol. (2004) 24, 3992-4003, Gonzalez H E. et al., ArchOtolaryngol Head Neck Surg. (2003) 129, 754-9, Sasaki H. et al., BreastCancer Res Treat. (2003) 77, 245-52), preeclampsia (Sasaki H. et al., AmJ Obstet Gynecol. (2002) 186, 103-8) as well as very slightly in normaltissue. Moreover, some periostin splice variants were shown to beexpressed in osteoblasts (Takeshita S. et al., Biochem J (1993) 294,271-8, Horiuchi K. et al., J. Bone Miner. Res. (1999) 14, 1239-49,Litvin J. et al., J Cell Biochem. (2004) 92, 1044-61, Katsuragi N. etal., Circulation (2004) 110, 1806-13).

As to functions of periostin, a periostin splice variant consisting of811 amino acids (corresponding to PN-2 in FIG. 1) (Horiuchi K. et al.,J. Bone Miner. Res. (1999) 14, 1239-49) and a periostin splice variantconsisting of 782 amino acids (Gillan L, et al., Cancer Res. (2002) 62,5358-64) were reported to have cell adhesive properties. In contrast, ithas been reported that a periostin splice variant consisting of 838amino acids (corresponding to PN-1 in FIG. 1) prevents heart fibroblastsfrom adhering to a plate coated with the periostin splicing variant,i.e., has no cell adhesive activity; the gene expression of theperiostin splice variant consisting of 838 amino acids (corresponding toPN-1 in FIG. 1) is significantly increased in heart failure model ratsas compared with normal rats; this variant is an aggravating factorinducing heart dilation; and that the survival rate was significantlyincreased by inhibition of the expression of this protein (Katsuragi N.et al., Circulation (2004) 110, 1806-13). Further, there is a report ofa prophylactic or therapeutic agent for heart failure, in which anantisense nucleotide against the periostin splice variant consisting of838 amino acids is used to suppress expression of the periostin splicingvariant (Republication WO02/020055).

In addition, the inventors of the present invention have reported aprophylactic or therapeutic agent for heart failure, which is based onthe following findings: the periostin splice variant consisting of 811amino acids (corresponding to PN-2 in FIG. 1) is involved in celladhesion whereas the periostin splice variant consisting of 838 aminoacids (corresponding to PN-1 in FIG. 1) has cell detachment activity; anantibody against an antigen composed of the Exon-17 sequence inhibitsthe cell detachment activity; and improved heart function was observedin acute myocardial infarction model animals (Japanese PatentApplication No. 2005-380009).

As to cancers, various reports have been issued on high level expressionof periostin in highly metastatic cancers [Erkan M. et al.Gastroenterology, 132(4), 1447-64 (2007) (pancreatic cancer),Siriwardena B S. et al. Br J Cancer, 95(10), 1396-403 (2006) (oralcancer), Baril P. et al. Oncogene, 26(14), 2082-94 (2007) (pancreaticcancer), Grigoriadis A. et al. Breast Cancer Res, 8(5), R56 (2006)(breast cancer), Kudo Y. et al. Cancer Res, 66(14), 6928-35 (2006) (headand neck cancer), Bao S. et al. Cancer Cell. 5(4), 329-39 (2004) (coloncancer), Shao R. et al., Mol Cell Biol. (2004) 24, 3992-4003 (breastcancer), Sasaki H. et al., Breast Cancer Res Treat. (2003) 77, 245-52(breast cancer), Sasaki H. et al. Cancer Lett., 72(1), 37-42 (2001)(thymic cancer), Sasaki H. et al. Cancer, 92(4), 843-8 (2001) (non-smallcell lung cancer), Gonzalez H E. et al., Arch Otolaryngol Head NeckSurg. (2003) 129, 754-9 (head and neck squamous cell carcinoma)]. Also,highly metastatic cancers are reported to express the transcriptionfactor Twist at high level (Thiery J P. et al. Nat Med. 10(8), 777-8(2004), Yang J, et al. Cell. 117(7), 927-39 (2004)) and receiveattention, but there is a report showing that Twist is also located inthe promoter region of periostin (Oshima A, et al. J Cell Biochem,86(4), 792-804 (2002)). In addition, it has been reported that the humanfetal kidney epithelial cell line 293T, which is carcinogenic andnon-metastatic, enhances its invasion ability when introduced with theperiostin gene (Yan W. et al. J Biol Chem., 281(28), 19700-8 (2006))).It has also been reported that a rat homolog of mouse periostin was lessexpressed in various cancer cells, introduction of the periostin geneinto bladder cancer cells inhibited invasion of the bladder cancercells, and introduction of the periostin gene into mouse melanomaB16-F10 cells inhibited their metastasis to lung (Kim C J, et al. Int JCancer, 117(1), 51-8 (2005)).

As shown above, it has been suggested that expression of the periostingene is related to the pathology of heart failure, but the relationshipbetween the structure of periostin splicing variants and heart failurehas been unknown.

Also, it has been suggested that expression of the periostin gene isrelated not only to the pathology of heart failure, but also to thepathology of cancer. However, it is unknown what function each splicevariant has on the progress of cancer condition.

Thus, we made an attempt to clarify the structure of periostin relatedto the pathology of heart failure by using antibodies.

As to periostin antibodies, there are reports of an antibody related tothe inhibition of chemotaxis of periostin (Lindner V. et al.,Arterioscler Thromb Vasc Biol. (2005) 25, 77-83) and an antibody havinginhibitory activity against periostin-induced cell growth (Tai I T, etal., Carcinogenesis (2005) 26, 908-15). However, there has been neithera report of antibodies showing the structure of a region responsible forcell adhesive activity of periostin nor a report showing the relationbetween the cell adhesive activity of periostin and diseases such asheart failure.

As to antibodies against periostin, there are reports of ananti-periostin antibody which inhibits periostin overexpression-enhancedmigration of mesenchymal cells (Lindner V. et al., Arterioscler ThrombVasc Biol. (2005) 25, 77-83) and an anti-periostin antibody whichinhibits periostin-induced growth and cell differentiation in colorectalcancer (Tai I T. et al., Carcinogenesis (2005) 26, 908-15).

SUMMARY OF THE INVENTION

The present invention aims to provide, e.g., a novel and effectiveprophylactic or therapeutic agent for heart failure, which enables animprovement in the quality of life and an improvement in long-term vitalprognosis. More specifically, the present invention aims to provide anantibody against a periostin isoform having anti-cell adhesive activity,specifically recognizing a site responsible for anti-cell adhesion. Thepresent invention also aims to provide a hybridoma producing theantibody, a method for producing the hybridoma, and a method forproducing the antibody by culturing the hybridoma. The present inventionfurther aims to provide a pharmaceutical composition comprising theantibody for preventing or treating a disease in which a periostinisoform having anti-cell adhesive activity is involved. The presentinvention further aims to provide a method for preventing or treating adisease in which a periostin isoform having anti-cell adhesive activityis involved, comprising administering the pharmaceutical composition toa patient, as well as a method for diagnosing the disease.

The present invention also aims to provide a novel cancer therapeuticagent which enables an improvement in the quality of life and animprovement in long-term vital prognosis and whose mechanism isdifferent from that of existing agents. The present invention furtheraims to provide a method and reagent for cancer diagnosis.

DETAILED DESCRIPTION OF THE INVENTION

We clarified that a periostin splicing variant having no cell adhesiveactivity (PN-1) has anti-cell adhesive activity, i.e., the activity ofdetachment of adhered cells. On the other hand, we also confirmed thatperiostin having cell adhesive activity (PN-2) has no anti-cell adhesiveactivity, i.e., does not detach adhered cells. Moreover, we noted adifference in structure and cell adhesive activity between periostinsplice variants having anti-cell adhesive activity (PN-1) and periostinshowing no anti-cell adhesive activity (PN-2), and considered thatdiseases related to periostin isoforms having anti-cell adhesiveactivity could be prevented or treated by inhibiting a regionspecifically present in the periostin splice variants having anti-celladhesive activity. In other words, we considered that inhibitors againstsaid region might be useful as prophylactic or therapeutic agents fordiseases related to periostin isoforms having anti-cell adhesiveactivity.

Analysis of periostin splice variants highly expressed during heartfailure revealed that the C-terminal domains in which the splicevariants are formed consist of exons 15 to 23; specifically rats havethe following variants (1) to (4):

(1) a variant retaining all the exons (called PN-1; consisting of 838amino acids shown as SEQ ID NO: 1; the cDNA sequence shown as SEQ ID NO:6),

(2) a variant lacking Exon-17 (called PN-2; consisting of 811 aminoacids shown as SEQ ID NO: 5; 27 amino acids (Exon-17) shown in SEQ IDNO: 3 are deleted from PN-1; the cDNA sequence shown as SEQ ID NO: 7),

(3) a variant lacking Exon-21 (called PN-3; consisting of 810 aminoacids),

(4) a variant lacking Exon-17 and Exon-21 (called PN-4; consisting of783 amino acids).

In addition to rats, mouse and human PN-1 and PN-2 were also found(mouse PN-1:SEQ ID NO: 8 (amino acid sequence), SEQ ID NO: 9 (cDNAsequence); mouse PN-2: SEQ ID NO: 10 (amino acid sequence), SEQ ID NO:11 (cDNA sequence); human PN-1: SEQ ID NO: 12 (cDNA sequence); humanPN-2: SEQ ID NO: 13 (amino acid sequence), SEQ ID NO: 14 (cDNAsequence)). Among them, PN-1 was highly expressed while PN-2 and PN-3were expressed to a lesser extent in rat cardiac hypertrophy tissue.Thus, we contemplated preparing an antibody specifically recognizing theamino acid residue part encoded by Exon-17 as an inhibitor against thatsite, which site is structurally different in PN-1 and PN-2 andexclusively found in PN-1.

In order to prepare an antibody, the material used as an immunogen mustbe hydrophilic, and if an antibody is to be prepared using a part of alarge polypeptide such as protein, the part used as an immunogen must beexposed on the surface of the protein to form an epitope site. Thus, inorder to examine the possibility of using the Exon-17 peptide chain asan antigen, an epitope search was initially performed using Accelryssoftware that is widely used in the field of bioinformatics Mac Vector7.2. It was judged from “Hydrophilicity”, “Surface Probability” and“Antigenicity” that TTKIITKLVEPKIKVIQGSLQPIIKTE (SEQ ID NO: 3) of theExon-17 region is mostly hydrophobic, suggesting that this region isvery unlikely to be exposed on the surface of the protein molecule andhas no immunogenicity, so it cannot be used to prepare an antibody, andtherefore, it was presumed that it would be difficult to use topractically prepare an antibody.

However, we ventured to prepare an antibody against the amino acidsequence encoded by Exon-17 on the assumption that the use of anantibody against a polypeptide region encoded by Exon-17 specificallyfound in PN-1 would be most suitable for specifically inhibitingfunctions of PN-1. A peptide consisting of 27 amino acids constituting apeptide encoded by the Exon-17 region was synthesized and used toimmunize rabbits, and the resulting serum was purified to give an IgGfraction, whereby an anti-Exon-17 peptide polyclonal antibody wasprepared. When periostin protein PN-1 was then added to an 80% confluentculture of heart fibroblasts, nearly 100% cell detachment (i.e.,anti-cell adhesive activity) was observed. Administration of theanti-Exon-17 peptide antibody to this experimental system inhibited thecell detachment mediated by periostin protein PN-1, showing that theanti-Exon-17 peptide antibody is an antibody having a neutralizingactivity against periostin protein PN-1. Then, acute myocardialinfarction model rats were prepared and weekly administration of theanti-Exon-17 peptide antibody was continued to show that heart dilationwas significantly inhibited 4 weeks after the preparation of the models,and that heart function was improved. It was also shown that theseproperties were sustained and cardiac fibrosis was inhibited even 8weeks after the preparation of the models. This indicates that theanti-Exon-17 peptide antibody is an antibody having the activity ofinhibiting heart dilation and cardiac fibrosis associated with theprogress of heart failure condition, and the activity of improving heartfunction, leading to accomplishment of the present invention.

As a solution to the problems described above, the present inventionprovides an antibody against a periostin isoform having anti-celladhesive activity specifically expressed in the heart during heartfailure or the like, particularly an antibody specifically recognizing asite responsible for anti-cell adhesion.

Based on these findings, the inventors of the present invention deducedthat the cell detachment effect (i.e., cell-releasing effect) of PN-1protein is related to the release of cancer cells from their primarytumor during the cancer metastasis processes mentioned above, andthereby facilitates cancer metastasis. Thus, they attempted to clarifythe relationship between PN-1 protein and the pathology of cancer.

The C-terminal domains in which periostin splice variants are formedconsist of exons 15 to 23; specifically rats have the following variants(1) to (4):

(1) a variant retaining all the exons (called PN-1; consisting of 838amino acids shown as SEQ ID NO: 1; the cDNA sequence shown as SEQ ID NO:6),

(2) a variant lacking Exon-17 (called PN-2; consisting of 811 aminoacids shown as SEQ ID NO: 5; 27 amino acids (Exon-17) shown in SEQ IDNO: 3 are deleted from PN-1; the cDNA sequence shown as SEQ ID NO: 7),

(3) a variant lacking Exon-21 (called PN-3; consisting of 810 aminoacids),

(4) a variant lacking Exon-17 and Exon-21 (called PN-4; consisting of783 amino acids).

In addition to rats, mouse and human PN-1 and PN-2 were also found(mouse PN-1: SEQ ID NO: 8 (amino acid sequence), SEQ ID NO: 9 (cDNAsequence); mouse PN-2: SEQ ID NO: 10 (amino acid sequence), SEQ ID NO:11 (cDNA sequence); human PN-1: SEQ ID NO: 12 (cDNA sequence); humanPN-2: SEQ ID NO: 13 (amino acid sequence), SEQ ID NO: 14 (cDNAsequence)).

Further, analysis of periostin splice variants highly expressed duringthe pathology of cancer revealed that PN-1 having the functions ofinhibiting cell adhesion and separating adhered cells is expressed atvery high level in tumor tissues of model mice for lung metastasis ofmouse melanoma B16-F10 cells or mouse 4T1 breast cancer cells whencompared to normal tissues, thus showing that PN-1 is expressed at avery high level during the pathology of cancer.

Then, the inventors of the present invention considered that a sitestructurally different between PN-1 having the function of inhibitingcell adhesion and PN-2 having the function of allowing cell adhesion,i.e., the Exon-17 region that is found only in PN-1 is a region relatedto the inhibition of cell adhesion, and further considered that wheninhibiting this Exon-17 region, it is possible to suppress the PN-1'sinhibitory function on cell adhesion and further inhibit cancermetastasis. Thus, the inventors contemplated preparing an antibodyspecifically recognizing a peptide part consisting of an amino acidsequence encoded by Exon-17 as an inhibitor against that site, whichsite is found in PN-1.

In order to prepare an antibody, the material used as an immunogen mustbe hydrophilic, and if an antibody is to be prepared using a part of alarge polypeptide such as protein, the part used as an immunogen must beexposed on the surface of the protein to form an epitope site. Thus, inorder to examine the possibility of using the Exon-17 peptide chain asan antigen, an epitope search was initially performed using Accelryssoftware Mac Vector 7.2 that is widely used in the field ofbioinformatics. It was judged from “Hydrophilicity”, “SurfaceProbability” and “Antigenicity” that TTKIITKLVEPKIKVIQGSLQPIIKTE (SEQ IDNO: 3) of the Exon-17 region is mostly hydrophobic, suggesting that thisregion is very unlikely to be exposed on the surface of the proteinmolecule and has no immunogenicity, so it cannot be used to prepare anantibody, and therefore, it was presumed that it would be difficult touse to practically prepare an antibody.

However, when a peptide consisting of 27 amino acids constituting apeptide encoded by the Exon-17 region was synthesized and used toimmunize rabbits, and the resulting serum was purified to give an IgGfraction, whereby an anti-Exon-17 peptide polyclonal antibody wasprepared, it was surprisingly possible to prepare an antibody against apeptide region encoded by Exon-17 specifically found in PN-1(hereinafter referred to as “anti-Exon-17 polyclonal antibody”) althoughthe peptide was presumed to be hydrophobic and non-immunogenic.

When PN-1 protein was then added to an 80% confluent culture of mousemelanoma B16-F10 cells or mouse 4T1 breast cancer cells, nearly 100%cell elimination (i.e., anti-cell adhesive activity) was observed.Administration of the anti-Exon-17 polyclonal antibody to thisexperimental system inhibited the cell elimination mediated by PN-1protein, showing that the anti-Exon-17 polyclonal antibody is anantibody having a neutralizing activity against PN-1 protein. Moreover,addition of the anti-Exon-17 polyclonal antibody to mouse melanomaB16-F10 cells or mouse 4T1 breast cancer cells inhibited cellproliferation, showing that the anti-Exon-17 polyclonal antibody alsohas an inhibitory effect on proliferation of mouse melanoma B16-F10cells or mouse 4T1 breast cancer cells.

When the anti-Exon-17 polyclonal antibody was then continued to beadministered once a week to model mice which will develop lungmetastasis when injected with mouse melanoma B16-F10 cells or mouse 4T1breast cancer cells into their soles, this antibody significantlyinhibited not only primary tumor growth, but also the metastasis rateand the number of metastasized colonies from primary tumor to lung,within 3 to 5 weeks after model preparation. Further, in mouse 4T1breast cancer cells, bone invasion of the breast cancer cells fromprimary tumor and bone destruction caused by bone invasion of cancerwere significantly inhibited. This suggested that a neutralizingantibody against PN-1 suppresses the PN-1's inhibitory effect onadhesion and has the potential to act as a novel therapeutic agent forinhibiting the growth of malignant melanoma or breast cancer cells, boneinvasion, bone destruction and lung metastasis. Moreover, when the samestudy was also performed on breast cancer 4T1 cells, not only primarytumor growth, but also the number of colonies metastasized from primarytumor to lung was significantly inhibited within 3 weeks afterpreparation of the model for lung metastasis of mouse 4T1 breast cancercells. This indicated that the anti-Exon-17 polyclonal antibody is anantibody having the activity of inhibiting primary tumor growthassociated with the progress of cancer condition, the activity ofinhibiting bone invasion, the activity of bone destruction caused bybone invasion of cancer, and having the activity of inhibitingmetastasis from primary tumor to lung. This showed that PN-1 protein hasthe function of causing the growth of primary tumor and metastasis fromprimary tumor during the pathology of cancer, leading to a finding thatthe progress of these cancer conditions can be inhibited by such anantibody against a peptide region encoded by Exon-17 specifically foundin PN-1.

Furthermore, the inventors of the present invention prepared amonoclonal antibody against the human periostin Exon-17 peptide chain(hereinafter referred to as “anti-Exon-17 monoclonal antibody”). Inexperiments using model mice for lung metastasis of mouse melanomaB16-F10 cells, administration of the anti-Exon-17 monoclonal antibodyshowed cancer growth inhibition and inhibitory effects on the metastasisrate and the number of metastasized colonies from primary tumor to lung.

These findings indicated that administration of the antibody against apeptide region encoded by Exon-17 specifically found in PN-1 (i.e.,anti-Exon-17 polyclonal antibody or anti-Exon-17 monoclonal antibody)allows inhibition of the progress of cancer condition, leading toaccomplishment of the present invention.

Namely, the present invention provides a pharmaceutical composition forcancer treatment, which comprises an antibody against periostin,particularly an antibody against a peptide region encoded by Exon-17.

Namely, the present invention includes the following aspects.

(1) An antibody against a periostin isoform having anti-cell adhesiveactivity, specifically recognizing a site responsible for anti-celladhesion of periostin, and having ability to neutralize anti-celladhesive activity of periostin.

(2) The antibody as defined in (1), wherein the site for responsible foranti-cell adhesion of periostin is the amino acid sequence encoded byExon-17 or by a part thereof.

(3) The antibody as defined in (2), wherein the amino acid sequenceencoded by Exon-17 or by a part thereof is one of the amino acidsequences selected from the group of SEQ IDs NO: 3, 4, 21, 22, 23, 24,26 and 34.

(4) The antibody as defined in (3), wherein the amino acid sequence isthe amino acid sequence of SEQ IDs NO: 3, 4, or 21.

(5) The antibody as defined in any one of (1) to (4), which is amonoclonal antibody.

(6) An antibody as defined in (5) produced by a hybridoma cell line FERMBP-10718.

(7) A hybridoma obtainable by a process comprising steps of:

immunizing a mammal with a peptide having one of the amino acidsequences selected from the group of SEQ IDs NO: 3, 4, and 21 or apeptide introduced cysteine residues into the N-terminus thereof; and

fusing an antibody-producing cell of the mammal with a myeloma cell.

(8) A hybridoma cell line FERM BP-10718.

(9) A method for producing an antibody as defined in (5), comprisingsteps of:

immunizing a mammal with a peptide having one of the amino acidsequences selected from the group of SEQ IDs NO: 3, 4, and 21 or apeptide introduced cysteine residues into the N-terminus thereof;

fusing an antibody-producing cell of the mammal with a myeloma cell; and

culturing the obtained hybridoma.

(10) The method as defined in (9), wherein the hybridoma is a hybridomacell line FERM BP-10718.

(11) A pharmaceutical composition comprising the antibody as defined inany one of (1) to (6).

(12) A method for preventing or treating a disease in which a periostinisoform having anti-cell adhesive activity is involved, comprisingadministering the antibody as defined in any one of (1) to (6) to apatient.

(13) The method as defined in (12), wherein the disease is heartfailure, myocardial infarction, heart dilation, cardiac hypertrophy,cardiac fibrosis, cardiomyopathy, myocarditis, valvular disease, cancer,aneurysm, arteriosclerosis, central neurodegenerative disease, renaldisease, rheumatoid arthritis, osteoporosis, pulmonary emphysema,pulmonary hypertension, chronic obstructive pulmonary disease (COPD),nephritis, pancreatitis, hepatitis, hepatic fibrosis or pulmonaryfibrosis.

(14) The method as defined in (13), wherein the disease is cancer.

(15) The method as defined in (14), wherein cancer is prevented ortreated by inhibiting cancer metastasis.

(16) The method as defined in (14), wherein cancer is prevented ortreated by inhibiting the growth of primary tumor.

(17) The method as defined in (14), wherein cancer is prevented ortreated by inhibiting the bone invasion of cancer or bone destructioncaused by bone invasion of cancer.

(18) The method as defined in any one of (14) to (17), wherein thecancer is malignant melanoma or breast cancer.

(19) A method for diagnosing a disease in which a periostin isoformhaving anti-cell adhesive activity is involved, comprising measuring theamount of the periostin isoform in a biological sample by using theantibody as defined in any one of (1) to (6).

(20) The method as defined in (19) wherein the antibody is a labeledantibody.

(21) The method as defined in (19) or (20), wherein the disease is heartfailure, myocardial infarction, heart dilation, cardiac hypertrophy,cardiac fibrosis, cardiomyopathy, myocarditis, valvular disease, cancer,aneurysm, arteriosclerosis, central neurodegenerative disease, renaldisease, rheumatoid arthritis, osteoporosis, pulmonary emphysema,pulmonary hypertension, chronic obstructive pulmonary disease (COPD),nephritis, pancreatitis, hepatitis, hepatic fibrosis or pulmonaryfibrosis.

(22) The method as defined in (21), wherein the disease is cancer.

(23) The method of as defined in (22), wherein cancer is prevented ortreated by inhibiting cancer metastasis.

(24) The method as defined in (22), wherein cancer is prevented ortreated by inhibiting the growth of primary tumor.

(25) The method as defined in (22), wherein cancer is prevented ortreated by inhibiting the bone invasion of cancer or bone destructioncaused by bone invasion of cancer.

(26) The method as defined in any one of (22) to (25), wherein thecancer is malignant melanoma or breast cancer.

(27) A method for detecting or quantifying a periostin isoform havinganti-cell adhesive activity in a sample by using the antibody as definedin any one of (1) to (6).

(28) A diagnostic reagent for a disease in which a periostin isoformhaving anti-cell adhesive activity is involved, comprising the antibodyas defined in any one of (1) to (6).

(29) The diagnostic reagent as defined in (28), wherein the disease isheart failure, myocardial infarction, heart dilation, cardiachypertrophy, cardiac fibrosis, cardiomyopathy, myocarditis, valvulardisease, cancer, aneurysm, arteriosclerosis, central neurodegenerativedisease, renal disease, rheumatoid arthritis, osteoporosis, pulmonaryemphysema, pulmonary hypertension, chronic obstructive pulmonary disease(COPD), nephritis, pancreatitis, hepatitis, hepatic fibrosis orpulmonary fibrosis.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a rat periostin splicing variant.

FIG. 2 is a diagram showing assay results of the anti-cell adhesiveproperties of rat PN-1 (Example 2).

FIG. 3 is a diagram showing assay results of the inhibition of rat PN-1activity by anti-rat Exon-17 peptide antibody (Example 3).

FIG. 4-1 is a diagram showing assay results of the inhibition of heartdilation by anti-rat Exon-17 peptide antibody 4 weeks after thepreparation of acute myocardial infarction models (Example 4: anteriorwall thickness, posterior wall thickness).

FIG. 4-2 is a diagram showing assay results of the inhibition of heartdilation by anti-rat Exon-17 peptide antibody 4 weeks after thepreparation of acute myocardial infarction models (Example 4:end-diastolic inner diameter, end-systolic inner diameter, heartfunction).

FIG. 4-3 is a diagram showing assay results of the inhibition of heartdilation by anti-rat Exon-17 peptide antibody 4 weeks after thepreparation of acute myocardial infarction models (Example 4: heartrate, infarction area).

FIG. 5-1 is a diagram showing assay results of the inhibition of heartdilation by anti-rat Exon-17 peptide antibody 8 weeks after thepreparation of acute myocardial infarction models (Example 4: anteriorwall thickness, posterior wall thickness).

FIG. 5-2 is a diagram showing assay results of the inhibition of heartdilation by anti-rat Exon-17 peptide antibody 8 weeks after thepreparation of acute myocardial infarction models (Example 4:end-diastolic inner diameter, end-systolic inner diameter, heartfunction).

FIG. 5-3 is a diagram showing assay results of the inhibition of heartdilation by anti-rat Exon-17 peptide antibody 8 weeks after thepreparation of acute myocardial infarction models (Example 4: heartrate, infarction area).

FIG. 6-1 is a diagram showing hemodynamics of model rats treated withanti-rat Exon-17 peptide antibody (Example 4: LVP, heart rate).

FIG. 6-2 is a diagram showing hemodynamics of model rats treated withanti-rat Exon-17 peptide antibody (Example 4: (+) dP/dt, (−) dP/dt).

FIG. 6-3 is a diagram showing hemodynamics of model rats treated withanti-rat Exon-17 peptide antibody (Example 4: SBP, DBP, LVEDP).

FIG. 7 is a diagram showing the results of histological analysis ofmodel rats treated with anti-rat Exon-17 peptide antibody (Example 4).

FIG. 8 is a diagram showing the minor axis diameters of myocardial cellsof model rats treated with anti-rat Exon-17 peptide antibody (Example4).

FIG. 9-1 is a diagram showing the results of gene expression analysis ofmodel rats treated with anti-rat Exon-17 peptide antibody (Example 4:G3PDH).

FIG. 9-2 is a diagram showing the results of gene expression analysis ofmodel rats treated with anti-rat Exon-17 peptide antibody (Example 4:ET-1/G3, Angiotensinogen/G3).

FIG. 9-3 is a diagram showing the results of gene expression analysis ofmodel rats treated with anti-rat Exon-17 peptide antibody (Example 4:α-MHC/G3, β-MHC/G3).

FIG. 9-4 is a diagram showing the results of gene expression analysis ofmodel rats treated with anti-rat Exon-17 peptide antibody (Example 4:Col-I/G3, Col-III/G3).

FIG. 9-5 is a diagram showing the results of gene expression analysis ofmodel rats treated with anti-rat Exon-17 peptide antibody (Example 4:TGF-β/G3, TNF-α/G3).

FIG. 10 is a diagram showing assay results of the anti-cell adhesiveproperties of human PN-1 (Example 15).

FIG. 11 is a diagram showing assay results of the inhibition of humanPN-1 activity by anti-human Exon-17 monoclonal antibody (Example 16).

FIG. 12-1 is a diagram showing assay results of the inhibition of heartdilation by anti-human Exon-17 monoclonal antibody 4 weeks after thepreparation of acute myocardial infarction models (Example 17: anteriorwall thickness, posterior wall thickness).

FIG. 12-2 is a diagram showing assay results of the inhibition of heartdilation by anti-human Exon-17 monoclonal antibody 4 weeks after thepreparation of acute myocardial infarction models (Example 17:end-diastolic inner diameter, end-systolic inner diameter, heartfunction).

FIG. 12-3 is a diagram showing assay results of the inhibition of heartdilation by anti-human Exon-17 monoclonal antibody 4 weeks after thepreparation of acute myocardial infarction models (Example 17: heartrate, infarction area).

FIG. 13 is a diagram showing assay results of the non-cell adhesiveproperties of rat PN-1 protein (Example 18).

FIG. 14 is a diagram showing assay results of the anti-cell adhesiveproperties of rat PN-1 protein (Example 19).

FIG. 15 is a diagram showing assay results of the inhibition of rat PN-1activity by anti-rat Exon-17 polyclonal antibody (Example 20).

FIG. 16A is a diagram showing assay results of the inhibition of mousemelanoma B16-F10 cell proliferation by anti-rat Exon-17 polyclonalantibody (Example 21).

FIG. 16B is a diagram showing assay results of the inhibition of mouse4T1 breast cancer cell proliferation by anti-rat Exon-17 polyclonalantibody (Example 21).

FIG. 17 is a diagram showing periostin expression in primary tumorinoculated with mouse melanoma B16-F10 cells and in normal tissue(Example 22).

FIG. 18 is a diagram showing assay results of the effect of anti-humanExon-17 monoclonal antibodies (No. 1 and No. 3) by using model mice forlung metastasis of mouse melanoma B16-F10 cells (Example 23).

FIG. 19 is a diagram showing assay results of the effect of anti-ratExon-17 polyclonal antibody by using model mice for lung metastasis ofmouse melanoma B16-F10 cells (Example 23: % increase in primary tumorsize, lung metastasis rate, number of metastasized colonies).

FIG. 20 is a diagram showing assay results of the effect of anti-humanExon-17 monoclonal antibody (No. 3) by using model mice for lungmetastasis of mouse melanoma B16-F10 cells (Example 23: % increase inprimary tumor size, lung metastasis rate, number of metastasizedcolonies).

FIG. 21 is a diagram showing assay results of the effect of anti-ratExon-17 polyclonal antibody by using model mice for lung metastasis ofmouse 4T1 breast cancer cells (Example 24: body weight change).

FIG. 22 is a diagram showing assay results of the effect of anti-ratExon-17 polyclonal antibody by using model mice for lung metastasis ofmouse 4T1 breast cancer cells (Example 24: tumor volume change inprimary tumor).

FIG. 23 is a diagram showing assay results of the effect of anti-ratExon-17 polyclonal antibody by using model mice for lung metastasis ofmouse 4T1 breast cancer cells (Example 24: comparison of bone area inbone destruction caused by bone invasion).

FIG. 24 is a diagram showing assay results of the effect of anti-ratExon-17 polyclonal antibody by using model mice for lung metastasis ofmouse 4T1 breast cancer cells (Example 24: comparison of number ofosteoclast in bone destruction caused by bone invasion).

FIG. 25 is a diagram showing assay results of the effect of anti-ratExon-17 polyclonal antibody by using model mice for lung metastasis ofmouse 4T1 breast cancer cells (Example 24: number of metastasizedcolonies at 3 weeks after inoculation of mouse 4T1 breast cancer cells).

THE MOST PREFERRED EMBODIMENTS OF THE INVENTION

In an embodiment, the present invention provides an antibody against aperiostin isoform having anti-cell adhesive activity. Periostin here isone of extracellular matrix proteins and is known to include some splicevariants, some of which are specifically expressed in the heart duringheart failure or the like. In the present invention, antibodies can beused as substances (i.e., as inhibitors) for inhibiting functions of aperiostin splicing variant specifically expressed in the heart duringheart failure or the like because of their high specificity, safety forhumans and for other reasons. In the present invention, an antibody canbe prepared against an antigen composed of a chemically synthesizedpeptide consisting of an amino acid sequence encoded by the Exon-17region of the C-terminal domain at which a splice variant is formed,though such a peptide can also be obtained from any source by enzymaticdigestion of periostin protein or by genetic engineering techniques.

In the present invention, the expression “having anti-cell adhesiveactivity” means having the action of separating or eliminating adheredcells. Likewise, the expression “having no anti-cell adhesive activity”means that adhered cells are neither detached nor peeled off. In thiscase, the cells maintain their adhered state. The presence or absence ofanti-cell adhesive activity may be determined by culturing cells (e.g.,heart fibroblasts) in a culture plate to allow the cells to adhere tothe culture plate, adding an assay sample and further culturing thecells, washing the plate to remove separated cells, staining theremaining cells, and confirming the state of the adhered cells.

In the present invention, periostin isoforms having anti-cell adhesiveactivity preferably include periostin isoforms consisting of an aminoacid sequence of SEQ ID NO: 1 (rat periostin PN-1, 838 amino acids), SEQID NO: 2 (human periostin PN-1, 836 amino acids having an N-terminalsignal sequence shorter by 2 amino acids than that of rat periostin), orSEQ ID NO: 8 (mouse periostin PN-1).

Regions responsible for the anti-cell adhesive activity of periostininclude, e.g., the site of Exon-17. Specific examples include an aminoacid residue part shown in SEQ ID NO: 3 of a periostin isoform having anamino acid sequence shown in SEQ ID NO: 1 (672-698 amino acids of SEQ IDNO: 1), an amino acid residue part shown in SEQ ID NO: 4 of a periostinisoform having an amino acid sequence shown in SEQ ID NO: 2 (670-696amino acids of SEQ ID NO: 2), and an amino acid residue part shown inSEQ ID NO: 21 of a periostin isoform having an amino acid sequence shownin SEQ ID NO: 8 (672-698 amino acids of SEQ ID NO: 8). Further examplesinclude an amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23,SEQ ID NO: 24 or SEQ ID NO: 26, as well as SEQ ID NO: 34 (an amino acidsequence shown in SEQ ID NO: 22 or SEQ ID NO: 23, as well as an aminoacid sequence consisting of the N-terminal 1st to 6th amino acidresidues of SEQ ID NO: 22 or SEQ ID NO: 23).

The expression “neutralizing the anti-cell adhesive activity ofperiostin” means that the action or activity of the above “regionresponsible for the anti-cell adhesive activity of periostin” isinhibited, and more specifically means that the action or activity ofperiostin is inhibited using, for example, an antibody capable ofspecifically recognizing the above site responsible for anti-celladhesive activity.

In an embodiment, antibodies of the present invention are monoclonalantibodies and polyclonal antibodies obtained by using the antigens asdescribed above. The “monoclonal antibodies” here refer to anymonoclonal antibody showing reactivity against the antigens describedabove, and the “monoclonal antibodies” include natural antibodiesobtained by immunizing mammals such as mice, rats, hamsters, guinea pigsor rabbits with the antigens, chimeric monoclonal antibodies (chimericantibodies) and humanized monoclonal antibodies (humanized antibodies;CDR-grafted antibodies) that can be prepared by using geneticrecombination techniques, as well as human monoclonal antibodies (humanantibodies) that can be prepared by using human antibody-producingtransgenic animals or the like. Antibodies of the present inventioninclude monoclonal antibodies having any isotype such as IgG (IgG1,IgG2, IgG3, IgG4), IgM, IgA, IgD or IgE, preferably IgG (IgG1, IgG2,IgG3, IgG4) or IgM.

When the peptides described above are to be used as antigens, they canbe used alone as antigens but also can be used for immunization byadsorption to a macromolecular material such as polyvinyl pyrrolidone,latex or polymethyl methacrylate, or coupling to a carrier protein suchas KLH (Keyhole Limpet Hemocyanin) or BSA (bovine serum albumin), andany method can be used. Generally, the peptides may preferably becoupled to a carrier protein by known methods (e.g., “New series ofDevelopment of Drugs, Vol. 14, Hirokawa Publishing Co., 1991”). Thepeptides are coupled to a carrier protein via cysteine residuesintroduced into the C- or N-terminus of the peptides so that thepeptides have directionality. Crosslinkers commonly used in the field ofthe art can be used so long as they are suitable for this purpose.Suitable crosslinkers includesuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (hereinafterabbreviated as “SMCC”) or 3-maleimidobenzoic acid-N-hydroxysuccinimideester (MBS). Monoclonal antibodies are prepared by culturing hybridomasprepared by the cell fusion method of Kohler and Milstein (G. Kohler etal Nature (1975) 256, 495-7) to secrete the antibodies and isolatingthem from the cultures. That is, a mammal is immunized with a peptidehaving an amino acid sequence encoded by Exon-17 or the like and thenantibody-producing cells of this animal are fused to myeloma cells togive hybridomas. Search for hybridomas producing antibodies binding toExon-17 is performed by e.g., an enzyme immunoassay (hereinafterabbreviated as “ELISA”) for hybridoma supernatants using a microplate onwhich the antigen has been immobilized. Animals to be immunized are notspecifically limited, but include various mammals such as mice, rats,guinea pigs, rabbits, sheep, goats, cats, dogs, etc. Among the animalslisted above, Balb/c mice are generally used for preparation ofmonoclonal antibodies because of ease of handling or for other reasons,but other strains of mice can also be used. The concentration of theantigen used for immunization here is selected to form sufficientamounts of antigenically stimulated lymphocytes. Preferably, 1-100 μg ofan antigen is diluted to an appropriate concentration with physiologicalsaline or the like and suspended in Freund's complete adjuvant orFreund's incomplete adjuvant or the like, and the suspension isadministered to an animal by intraperitoneal or subcutaneous injectionor other means. Administration is performed once to several times every2-4 weeks. The final immunization is normally performed by administeringa solution of 1-100 μg of the antigen in physiological saline byintravenous or subcutaneous injection or other means. Several days afterthe final immunization, antibody-producing cells such as lymphocytes,preferably spleen cells or lymph node cells, are removed from theimmunized animal for cell fusion. Cell fusion using spleen cells asantibody-producing cells is explained below, though antibody-producingcells other than spleen cells can also be used for cell fusion. Spleencells prepared from the spleen aseptically removed 3-4 days after thefinal immunization are fused to appropriate myeloma cells in thepresence of a fusion promoter. The myeloma cells used for fusion may bederived from mammals, but generally those derived from the same speciesas the animal used for immunization. Various cell lines are alreadyknown, for example, SP2/0-Ag14(SP2) [Nature, 276, 269 (1978)],NS-1-Ag4/1(NS-1), P3-X63Ag8U.1(P3U1) [Curr. Top. Microbiol. Immunol. 81,1-7 (1978): available from ATCC under ATCC No. CRL-1597],P3-NS1-1-Ag4-1, P3-X63Ag8(P3), FO, X63Ag8.653(X63.653),210.RCY3.Ag1.2.3, S194/5XXO.BU1, SKO-007, GM15006TG-A12 and the like arepreferably used for mice, and Y3.Ag1.2.3 and the like are preferablyused for rats. Preferred fusion promoters include polyethylene glycol(PEG) having a molecular weight of 1000-6000 and Sendai virus (HVJ).Generally, the ratio of spleen cells and myeloma cells during fusion ispreferably 10:1-2:1.

Hybridomas can be separated from fused cells by culturing of a mixtureof unfused spleen cells, unfused myeloma cells and fused cells in aselective medium inhibiting the survival of unfused myeloma cells for anappropriate period until unfused cells die (about 1 week). The selectivemedium may be e.g., HAT medium (medium containing hypoxanthine,aminopterin and thymidine). In this selective medium, unfused myelomacells die, and unfused spleen cells die after a certain period of time(after about 1 week) because they are non-tumorous cells, so thathybridomas can be obtained by selecting viable cells. Hybridomasproducing desired antibodies can be obtained by searching of strainsproducing the desired antibodies and cloning of the strains to preparemonoclonal antibodies by standard limiting dilution. Thus obtainedhybridomas producing monoclonal antibodies of the present invention cangrow in media suitable for their growth and can be readily stored indeep freezers or liquid nitrogen. Thus obtained hybridomas can produceantibodies by growing them in nutrient media or in the abdominal cavityof a mammal, and the produced antibodies can be purified from culturesupernatants or the ascites fluid or serum of the mammal. As an exampleof the hybridomas of the present invention, a hybridoma can be used thatwas deposited under FERM BP-10718 on Nov. 1, 2006 with the InternationalPatent Organism Depositary, the National Institute of AdvancedIndustrial Science and Technology. Purification of the antibodies can beperformed by standard isolation/purification methods such ascentrifugation, dialysis, salting out with ammonium sulfate or the like,ion exchange chromatography using a DEAE column or the like, gelfiltration, affinity chromatography, etc. The isotypes and subclasses ofthus obtained monoclonal antibodies can be determined using anidentification method such as Ouchterlony assay, ELISA, or RIA.Ouchterlony assay is convenient but requires a concentration operationif the monoclonal antibody concentration is low. When ELISA or RIA isused, however, the isotypes and subclasses of the monoclonal antibodiescan be identified by direct reaction of the culture supernatant with anantigen-adsorbed solid phase and by use of antibodies corresponding tovarious immunoglobulin isotypes and subclasses as secondary antibodies.More convenient methods employ commercially available identificationkits (e.g., Mouse Typer kit; Bio-Rad) or the like. The quantification ofprotein can be performed by the Folin-Lowry method and calculation fromthe absorbance at 280 nm [1.4 (OD280)=1 mg/ml immunoglobulin]. Thusobtained monoclonal antibodies of the present invention specificallyrecognize an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2,an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, aperiostin isoform (PN-1) having an amino acid sequence shown in SEQ IDNO: 34, a peptide consisting of an amino acid sequence shown in SEQ IDNO: 3 or SEQ ID NO: 4, or a peptide having an amino acid sequence shownin SEQ ID NO: 34. Preferably, the monoclonal antibodies of the presentinvention can specifically recognize and bind to a peptide consisting ofthe amino acid sequence YTTKIITKVV (SEQ ID NO: 26), i.e., a peptideconsisting of an amino acid sequence covering from the −1st tyrosine tothe 9th valine from the N-terminus of the amino acid sequence of thehuman periostin Exon-17 peptide chain (SEQ ID NO: 4), and a peptideconsisting of an amino acid sequence covering from the 669th tyrosine tothe 679th valine from the N-terminus of the amino acid sequence of humanperiostin PN-1 (SEQ ID NO: 2). Namely, the monoclonal antibodies of thepresent invention can specifically recognize an amino acid sequence site(TTKIITKVV; SEQ ID NO: 22), or a part thereof, which covers from theN-terminal threonine to the 9th valine of the amino acid sequence of thehuman periostin Exon-17 peptide chain (SEQ ID NO: 4). More preferably,the monoclonal antibodies of the present invention can recognize andbind to a peptide comprising alanine substitutions at the 1st and the8-10th amino acids from the N-terminus of a peptide consisting of anamino acid sequence (YTTKIITKW; SEQ ID NO: 26) covering from the −1sttyrosine to the 9th valine from the N-terminus of the human periostinExon-17 peptide chain (SEQ ID NO: 4). Namely, the monoclonal antibodiesof the present invention can specifically recognize at least an aminoacid sequence site (SEQ ID NO: 34), or a part thereof, which covers fromthe 1st threonine to the 6th threonine from the N-terminus of the aminoacid sequence of the human periostin Exon-17 peptide chain (SEQ ID NO:4) or the amino acid sequence of the rat periostin Exon-17 peptide chain(SEQ ID NO: 3). Moreover, the monoclonal antibodies of the presentinvention have the activity of suppressing or inhibiting the anti-celladhesive properties of human periostin-1 protein, i.e., neutralizing theanti-cell adhesive properties of human periostin-1 protein. Furthermore,the monoclonal antibodies of the present invention can suppress theheart dilation, cardiac hypertrophy and cardiac fibrosis induced duringheart failure or the like to improve heart function. Moreover, themonoclonal antibodies for use in the present invention have the activityof suppressing or inhibiting the PN-1 protein's effects of inhibitingcancer cell adhesion and separating adhered cells, i.e., the activity ofneutralizing the PN-1 protein's effects of inhibiting cancer celladhesion and separating adhered cells. The monoclonal antibodies for usein the present invention further have inhibitory activities on thegrowth of primary tumor, the bone invasion of cancer cells, the bonedestruction caused by bone invasion of cancer cells, and metastasis.

When polyclonal antibodies are used as antibodies of the presentinvention, the polyclonal antibodies can be obtained by standard methodssuch as the method described in “New Lecture on Biochemical Experiments,12, edited by the Japanese Biochemical Society, Tokyo Kagaku Dozin,1992”. Animals to be immunized are not specifically limited, but includehorses, goats, sheep, rabbits, guinea pigs, mice, chickens, etc. When arabbit is to be immunized, an antigen is diluted to an appropriateconcentration with physiological saline or the like and suspended inFreund's complete adjuvant, Freund's incomplete adjuvant or aluminumhydroxide adjuvant or the like, and the suspension is injected at a doseof 10-1000 μg per animal followed by 1-3 booster injections after 2-4weeks to give antisera. Multi-site subcutaneous injection is preferred.Preparation of polyclonal antibodies from antisera can be performed bythe method as described for the purification of monoclonal antibodies.Thus obtained polyclonal antibodies of the present inventionspecifically recognize an amino acid sequence shown in SEQ ID NO: 1 orSEQ ID NO: 2, an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:4, a periostin isoform (PN-1) having an amino acid sequence shown in SEQID NO: 34, a peptide consisting of an amino acid sequence shown in SEQID NO: 3 or SEQ ID NO: 4, or a peptide having an amino acid sequenceshown in SEQ ID NO: 34. Preferably, the polyclonal antibodies of thepresent invention can specifically recognize and bind to a peptideconsisting of the amino acid sequence YTTKIITKVV (SEQ ID NO: 26), i.e.,a peptide consisting of an amino acid sequence covering from the −1sttyrosine to the 9th valine from the N-terminus of the amino acidsequence of the human periostin Exon-17 peptide chain (SEQ ID NO: 4),and a peptide consisting of an amino acid sequence covering from the669th tyrosine to the 679th valine from the N-terminus of the amino acidsequence of human periostin PN-1 (SEQ ID NO: 2). Namely, the polyclonalantibodies of the present invention can specifically recognize an aminoacid sequence site (TTKIITKVV; SEQ ID NO: 22), or a part thereof, whichcovers from the N-terminal threonine to the 9th valine of the amino acidsequence of the human periostin Exon-17 peptide chain (SEQ ID NO: 4).More preferably, the polyclonal antibodies of the present invention canrecognize and bind to a peptide comprising alanine substitutions at the1st and the 8-10th amino acids from the N-terminus of a peptideconsisting of an amino acid sequence (YTTKIITKVV; SEQ ID NO: 26)covering from the −1st tyrosine to the 9th valine from the N-terminus ofthe human periostin Exon-17 peptide chain (SEQ ID NO: 4). Namely, thepolyclonal antibodies of the present invention can specificallyrecognize at least an amino acid sequence site (SEQ ID NO: 34), or apart thereof, which covers from the 1st threonine to the 6th threoninefrom the N-terminus of the amino acid sequence of the human periostinExon-17 peptide chain (SEQ ID NO: 4) or the amino acid sequence of therat periostin Exon-17 peptide chain (SEQ ID NO: 3). Moreover, thepolyclonal antibodies of the present invention have the activity ofsuppressing or inhibiting the anti-cell adhesive properties of humanperiostin PN-1 protein, i.e., neutralizing the anti-cell adhesiveproperties of human periostin PN-1 protein. Furthermore, the polyclonalantibodies of the present invention can suppress the heart dilation,cardiac hypertrophy and cardiac fibrosis induced during heart failure orthe like to improve heart function. Moreover, the polyclonal antibodiesfor use in the present invention have the activity of suppressing orinhibiting the PN-1 protein's activity of inhibiting cancer celladhesion and separating adhered cells, i.e., the activity ofneutralizing the PN-1 protein's activity of inhibiting cancer celladhesion and separating adhered cells. The polyclonal antibodies for usein the present invention further have inhibitory activities on thegrowth of primary tumor, the bone invasion of cancer cells, the bonedestruction caused by bone invasion of cancer cells, and metastasis ofcancer cells.

Preparation of Humanized Antibodies

Immunoglobulin G (hereinafter simply referred to as “IgG”) consists oftwo light polypeptide chains having a molecular weight of about 23000(hereinafter referred to as “light chain”) and two heavy polypeptidechains having a molecular weight of about 50000 (hereinafter referred toas “heavy chain”). The heavy and light chains both have a repeatingstructure of conserved amino acid sequence regions consisting of about110 residues, which constitute a basic unit of three-dimensionalstructure of IgG (hereinafter referred to as “domain”). The heavy andlight chains consist of 4 and 2 successive domains, respectively. Inboth of the heavy and light chains, the amino acid sequence of the aminoterminal domain is more variable between antibody molecules than that ofthe other domains, and this domain is called variable domain(hereinafter referred to as “V domain”). At the amino terminus of IgG,the V domains of the heavy and light chains are complementarilyassociated to form a variable region. In contrast, the remaining domainscollectively form a constant region. The constant region has a sequencecharacteristic of each animal species, e.g., the constant region ofmouse IgG differs from the constant region of human IgG so that mouseIgG is recognized as foreign matter by the human immune system,resulting in a Human Anti Mouse Antibody (hereinafter referred to as“HAMA”) response (Schroff R W, et al. Cancer Res. (1985) 45, 879-85).Thus, mouse antibodies cannot be repeatedly administered to humans. Inorder to administer such antibodies to humans, the antibody moleculesmust be modified to prevent HAMA response while maintaining thespecificity of the antibodies. According to the results of X-raycrystallography, such a domain is generally in the form of an ellipticcylindrical structure formed of two antiparallel beta sheets consistingof 3 to 5 beta-chains. In the variable region, three loops for each ofthe V domains of the heavy and light chains are assembled to form anantigen-binding site. These loops are called complementarity determiningregions (hereinafter referred to as “CDRs”), which are most variable inamino acid sequence. The remaining parts of the variable region otherthan the CDRs serve to maintain the structures of the CDRs and arecalled “framework”. Kabatt et al. collected a number of primarysequences of heavy and light chain variable regions and prepared a tableclassifying the primary sequences into CDRs and frameworks on the basisof sequence conservation (Kabatt et al. SEQUENCES OF IMMUNOLOGICALINTEREST, 5th edition, NIH publication, No. 91-3242, E.A.). Theframeworks were further classified into a plurality of subgroups havingcommon amino acid sequence patterns. The presence of a consensusframework between human and mouse sequences was also found. Such studieson structural features of IgG led to the development of the processesfor preparing humanized antibodies described below. At an early stage ofthe studies, chimeric antibodies having a variable region from a mouseantibody fused to a constant region from a human antibody were proposed(Morrison S L. et al Proc Natl Acad Sci USA. (1984)81, 6851-5). However,such chimeric antibodies may induce a HAMA response, especially whenthey are administered for a long term, because they still contain manynon-human amino acid residues (Begent et al., Br. J. Cancer, (1990)62,487).

A method for further reducing amino acid residues derived from anon-human mammal that may induce a HAMA response to humans bytransferring only the CDRs into a human antibody was proposed (Peter Tet al. Nature, (1986) 321, 522-5), but grafting of only the CDRs wasnormally insufficient to maintain immunoglobulin activity against theantigen. On the other hand, Chothia et al. used X-ray crystallographicdata in 1987 to find that (a) the amino acid sequences of the CDRscontain a site directly binding to the antigen and a site maintainingthe structures of the CDRs, and possible three-dimensional structures ofthe CDRs are classified into multiple typical patterns (canonicalstructures), and that (b) the classes of the canonical structures aredetermined by not only the CDRs but also the types of amino acids atspecific locations on the framework (Chothia C. et al. J. Mol. Biol.(1987)196, 901-17). Based on this finding, a document suggested thatwhen CDR grafting is used, amino acid residues on a part of theframework should also be grafted into a human antibody in addition tothe CDR sequences (JPA No. HEI-4-502408). Generally, an antibody derivedfrom a non-human mammal having CDRs to be grafted is defined as “donor”,and a human antibody into which the CDRs are grafted is defined as“acceptor”, and considerations in CDR grafting are to conserve thestructures of the CDRs to the extent possible to maintain the activityof the immunoglobulin molecule. To achieve this object, two pointsshould be kept in mind, i.e. (a) which subgroup of acceptor should beselected, and (b) which amino acid residue should be selected from theframework of the donor.

Queen et al. proposed methods for designing immunoglobulins wherein anamino acid residue in the framework of a donor is grafted into anacceptor in addition to the CDR sequences when at least one of thefollowing criteria is satisfied (JPA No. HEI-4-502408):

(a) the amino acid in the framework region of the acceptor is rare forthat position and the corresponding amino acid in the donor is commonfor that position;

(b) the amino acid is immediately adjacent to one of the CDRs; or

(c) the amino acid is predicted to have a side chain atom within about 3angstroms of the CDRs in a three-dimensional immunoglobulin model and tobe capable of interacting with the antigen or with the CDRs of thehumanized antibody.

The DNA encoding the heavy or light chain of an anti-Exon-17 monoclonalantibody of the present invention can be obtained by preparation of mRNAfrom hybridoma cells producing the anti-Exon-17 monoclonal antibody,conversion of the mRNA into cDNA by reverse transcriptase and thenisolation of the DNA encoding the heavy or light chain of the antibody.

Preparation of Human Antibodies

As used herein, the “human antibody” or “human immunoglobulin” means animmunoglobulin in which all the regions constituting the immunoglobulinincluding heavy chain variable regions (VH) and heavy chain constantregions (CH) as well as light chain variable regions (VL) and lightchain constant regions (CL) are derived from genes encoding a humanimmunoglobulin. In other words, it means an antibody in which the heavychain is derived from a human immunoglobulin heavy chain gene and thelight chain is derived from a human immunoglobulin light chain gene.Human antibodies can be prepared by standard methods, e.g., byimmunization of a transgenic animal prepared by integration of at leasta human immunoglobulin gene into the locus of a non-human mammal such asa mouse with an antigen, in the same manner as described above for thepreparation of monoclonal antibodies. For example, transgenic miceproducing human antibodies can be prepared by the methods described inprior documents (Mendez M J et al. Nature Genetics (1997)15, 146-56,Green L L et al. Nature Genetics (1994)7, 13-21, JPA HEI-4-504365;International Publication No. WO94/25585; Nikkei Science, June, pp.40-50, 1995; Nils Lonberg et al. Nature (1994) 368, 856-9, and JPA No.HEI-6-500233).

Antibodies used in the present invention are not limited to wholeantibody molecules and may be antibody fragments or derivatives as longas they can neutralize the activity of a periostin isoform havinganti-cell adhesive activity.

Antibody fragments include, for example, Fab, F(ab′)₂, Fv, single chainantibody (scFv), disulfide-stabilized antibody (dsFv), a CDR-containingpeptide, etc.

Among the antibody fragments of the present invention, Fab, F(ab′)₂ andthe like can be obtained by treating an antibody inhibiting theanti-cell adhesive activity of periostin with a proteolytic enzyme suchas papain or pepsin, or alternatively, can be prepared by constructing agene encoding the resulting antibody fragment and introducing thisconstruct into an expression vector, followed by expression in anappropriate host cell.

Among the antibody fragments of the present invention, scFv can beprepared by linking together an H chain V region and an L chain V regionfrom an antibody inhibiting the anti-cell adhesive activity of periostinby using an appropriate peptide linker or the like. Alternatively, scFvcan be prepared by constructing a DNA segment encoding the entiresequences or desired amino acid sequences of a gene encoding an H chainor H chain V region from the above antibody and a gene encoding an Lchain or L chain V region from the antibody, and introducing thisconstruct into an expression vector, followed by expression in anappropriate host cell.

Among the antibody fragments of the present invention, dsFv is anantibody fragment in which polypeptides modified to replace one aminoacid residue by a cysteine residue in both H and L chain V regions froman antibody inhibiting the anti-cell adhesive activity of periostin arelinked together between these cysteine residues via a disulfide linkage.An amino acid residue to be replaced by a cysteine residue can beselected by stereostructural estimation of the antibody. dsFv can beprepared by constructing a DNA segment encoding the entire sequence or adesired amino acid sequence of a gene encoding the antibody fragment,and introducing this construct into an expression vector, followed byexpression in an appropriate host cell.

Among the antibody fragments of the present invention, a CDR-containingpeptide comprises at least one or more CDR regions selected from CDRregions in H or L chains of an antibody inhibiting the anti-celladhesive activity of periostin. Also, multiple CDR regions may be linkedtogether by techniques using an appropriate peptide linker or the like.The CDR-containing peptide may also be prepared by constructing a DNAsegment encoding the entire sequence or a desired amino acid sequence ofa gene encoding the peptide, and introducing this construct into anexpression vector, followed by expression in an appropriate host cell.Alternatively, the CDR-containing peptide can also be prepared bychemical synthesis such as Fmoc or tBoc method.

In the present invention, it is also possible to use derivatives of theabove antibodies or antibody fragments, which are modified to have aprotein or low-molecular compound bound thereto. These modifications maybe accomplished by known techniques.

In an embodiment, the antibodies, antibody fragments or derivatives ofthe present invention can be used to prevent or treat diseases in whicha periostin isoform having anti-cell adhesive activity is involved.“Diseases in which a periostin isoform having anti-cell adhesiveactivity is involved” refer to diseases during which a gene of aperiostin isoform having anti-cell adhesive activity is highly expressedand the production of a protein encoded by the gene is increased. Theyalso refer to diseases whose pathology is exacerbated by an increase inthe gene or protein. Such diseases in which a periostin isoform havinganti-cell adhesive activity is involved are not specifically limited,but include heart failure, myocardial infarction, heartenlargement(dilation), cardiac hypertrophy, cardiac fibrosis,cardiomyopathy, myocarditis, valvular disease, cancers, aneurysms,arteriosclerosis, central neurodegenerative disease, renal diseases,rheumatoid arthritis, osteoporosis, pulmonary emphysema, pulmonaryhypertension, chronic obstructive pulmonary disease (COPD), (acute andchronic) nephritis, (acute and chronic) pancreatitis, (acute andchronic) hepatitis, hepatic fibrosis or pulmonary fibrosis. Cancers towhich the antibodies of the present invention can be applied include,but not limited to, cancers of breast, large intestine, lung, malignantmelanoma, bone, pancreas, stomach, skin, uterus, ovary, rectum, colon,uterus, fallopian tube, esophagus, small intestine, thyroid,parathyroid, adrenal gland, prostate, bladder and kidney, especiallycancers of breast, large intestine, lung and malignant melanoma.

The present invention also provides a diagnostic reagent for a disease(e.g., heart failure) in which a periostin isoform having anti-celladhesive activity is involved, prepared by labeling of an antibody asdescribed above with a marker. Markers that can be used here includeenzymes, radioisotopes, fluorescent dyes, etc. The enzymes used here arenot specifically limited so long as they satisfy criteria such as highturnover number, stability even after conjugation, and the ability tospecifically react with their substrates to develop color, etc., andenzymes used in standard enzyme immunoassays (EIA) can be used. Examplesof preferred enzymes include peroxidases, β-galactosidases, alkalinephosphatases, glucose oxidase, acetylcholine esterase,glucose-6-phosphate dehydrogenase, malate dehydrogenase, etc. Enzymeinhibitors and coenzymes and the like can also be used.

Conjugation of these enzymes and antibodies can be performed by knownmethods using crosslinkers such as maleimide compounds. Substrates thatcan be used are known materials, selected depending on the enzymes used.For example, when the enzyme used is a peroxidase,3,3′,5,5′-tetramethylbenzidine can be used, or when the enzyme used isan alkaline phosphatase, paranitrophenol or the like can be used.Radioisotopes that can be used as markers include those used in thestandard radioimmunoassay (RIA) such as 125I and 3H. Fluorescent dyesthat can be used are those used in the standard fluoroimmunoassay suchas fluorescence isothiocyanate (FITC) and tetramethyl rhodamineisothiocyanate (TRITC). The present diagnostic reagent can also be usedas immunohistological staining capable of specifically staining affectedinterstitial tissue of the heart. When it is labeled with aradioisotope, it can also be used to image the lesion during heartfailure by internal administration.

The present invention also provides a method for detecting orquantifying a periostin isoform having anti-cell adhesive activity in abiological sample obtained by preparing serum from human or animalblood, i.e., in serum, comprising the use of the antibody, antibodyfragment or derivative of the present invention. The present inventionfurther provides a method for diagnosing a disease (e.g., heart failure)in which a periostin isoform having anti-cell adhesive activity isinvolved, comprising detection or quantification of the periostinisoform. In the present method, a periostin isoform having anti-celladhesive activity can be detected by a so-called sandwich ELISA(Enzyme-linked immunosorbent assay). When the diagnostic kit of thepresent invention is used, a sample is initially contacted with a plateon which a primary anti-periostin antibody has been immobilized to forma complex, and a secondary anti-periostin antibody labeled with a markeris bound to this complex, and then the signal intensity of the marker inthis ternary complex is measured, whereby a periostin isoform havinganti-cell adhesive activity can be detected or quantified. Inparticular, since a periostin isoform having anti-cell adhesive activityis a splicing variant which is specifically expressed during thepathology of heart failure or the like, the pathology in heart failureor the like can be diagnosed by monitoring its production.

In this way, an antibody of the present invention can be used here asthe secondary antibody by labeling of the antibody.

Pharmaceutical compositions comprising the antibodies, antibodyfragments or derivatives of the present invention as active ingredientsmay be prepared using carriers and/or excipients or other additives,which are used in standard formulation techniques.

The active ingredients of the pharmaceutical compositions according tothe present invention are preferably administered in admixture withknown pharmacologically acceptable carriers, excipients, diluents or thelike by any administration mode commonly used for pharmaceuticalpreparations, for example, by the oral or parenteral (e.g., intravenous,intramuscular or subcutaneous) route. For example, the pharmaceuticalcompositions of the present invention can be prepared by appropriatelymixing the active ingredients with physiologically acceptable carriers,flavors, excipients, stabilizers, diluents, emulsifiers, solutions,suspensions, syrups or the like, and can be used in the form of tablets,powders, granules, solutions or the like. Additives which can beincorporated into tablets or the like include, for example, binders suchas gelatin and lubricants such as corn starch. Tablets may also becoated with a sugar coating or a gastric or enteric film. In the dosageform of capsules, the above compositions can further comprise liquidcarriers. Injectable sterile compositions can also be prepared byapplying standard formulae. Injectable aqueous vehicles include isotonicsolutions containing glucose and the like, which may be used incombination with appropriate solubilizers such as polyethylene glycol,etc. The compositions may also be incorporated with buffers,stabilizers, preservatives, antioxidants, soothing agents and the like.For oral administration, when the active ingredients are likely to bedecomposed in the digestive tract, the compositions may be administeredorally as formulations that are resistant to decomposition in thedigestive tract, for example, as microcapsules encapsulating the activeingredients within liposomes. It is also possible to use otheradministration modes intended for absorption through mucous membranesother than the digestive tract, including rectal, intranasal, sublingualand transpulmonary routes. In this case, the compositions can beadministered in the form of suppositories, nose drops, sublingualtablets, transpulmonary agents or the like.

When the pharmaceutical compositions of the present invention are usedfor therapeutic purposes, their dosage is set at a therapeuticallyeffective dosage, which varies depending on, e.g., an age and a bodyweight of a subject to which the composition is to be administered, aseverity of symptoms and a route of administration, and thusadministration is determined on an individual basis. In general, thedaily adult dosage for oral administration is about 0.1 to 1000 mg,given as a single dose or in divided doses. For continuous intravenousadministration, the compositions can be administered in the range of0.01 μg/kg/min to 1.0 μg/kg/min, desirably 0.025 μg/kg/min to 0.1μg/kg/min.

Pharmaceutical Composition for Cancer Treatment

The present invention is also directed to a pharmaceutical compositionfor cancer treatment, which comprises an anti-periostin antibodyrecognizing a periostin splice variant having anti-cell adhesiveproperties (PN-1 protein).

The pharmaceutical composition of the present invention can be used forcancer treatment or diagnosis. Cancers to be treated or diagnosed by thepresent invention include, but not limited to, malignant melanoma, aswell as cancers of breast, large intestine, lung, bone, pancreas,stomach, skin, uterus, ovary, rectum, colon, uterus, fallopian tube,esophagus, small intestine, thyroid, parathyroid, adrenal gland,prostate, bladder and kidney. The pharmaceutical composition of thepresent invention can be particularly applied to highly metastaticcancers such as malignant melanoma, breast cancer, large intestinecancer or lung cancer.

The pharmaceutical composition of the present invention has inhibitoryeffects on the growth of primary tumor, on the bone invasion of cancercells, on the bone destruction caused by bone invasion of cancer cells,and on cancer metastasis. Thus, cancer treatment can be achieved byinhibiting the growth of primary tumor, the bone invasion of cancercells, the bone destruction caused by bone invasion of cancer cells, orby inhibiting cancer metastasis.

The pharmaceutical composition of the present invention may be preparedusing carriers and/or excipients or other additives, which are used instandard formulation techniques.

The antibody serving as an active ingredient in the pharmaceuticalcomposition of the present invention is preferably administered inadmixture with known pharmacologically acceptable carriers, excipients,diluents or the like by any administration mode commonly used forpharmaceutical preparations, especially antibody drugs, for example, viathe intravenous, subcutaneous, intracutaneous, intramuscular,intraperitoneal or oral route.

The pharmaceutical composition of the present invention can be preparedby appropriately mixing the active ingredient with physiologicallyacceptable carriers, flavors, excipients, stabilizers, diluents,emulsifiers, solutions, suspensions, syrups or the like, and can be usedin the form of tablets, powders, granules, solutions or the like.Additives which can be incorporated into tablets or the like include,for example, binders such as gelatin and lubricants such as corn starch.Tablets may also be coated with a sugar coating or a gastric or entericfilm. In the dosage form of capsules, the above composition can furthercomprise liquid carriers. Injectable sterile compositions can also beprepared by applying standard formulae. Injectable aqueous vehiclesinclude isotonic solutions containing glucose and the like, which may beused in combination with appropriate solubilizers such as polyethyleneglycol, etc. The compositions may also be incorporated with buffers,stabilizers, preservatives, antioxidants, soothing agents and the like.For oral administration, when the active ingredient is likely to bedecomposed in the digestive tract, the composition may be administeredorally as a formulation that is resistant to decomposition in thedigestive tract, for example, as microcapsules encapsulating the activeingredient within liposomes. It is also possible to use otheradministration modes intended for absorption through mucous membranesother than the digestive tract, including rectal, intranasal, sublingualand transpulmonary routes. In this case, the composition can beadministered in the form of suppositories, nose drops, sublingualtablets, transpulmonary agents or the like.

When the pharmaceutical composition of the present invention is used fortherapeutic purposes, its dosage is set at a therapeutically effectivedosage, which varies depending on, e.g., an age and a body weight of asubject to which the composition is to be administered, a severity ofsymptoms and a route of administration, and thus administration isdetermined on an individual basis. In general, the daily adult dosagefor oral administration is about 0.1 to 1000 mg, given as a single doseor in divided doses. For continuous intravenous administration, thecomposition can be administered in the range of 0.01 μg/kg/min to 1.0μg/kg/min, desirably 0.025 μg/kg/min to 0.1 μg/kg/min.

Diagnostic Method

The present invention is also directed to a method for cancer diagnosis,which comprises measuring the amount of a periostin isoform having apeptide region encoded by Exon-17 in a biological sample, for example, aserum sample prepared from human or animal blood by using an antibodyfor use in the present invention. In the present method, a periostinisoform having a peptide region encoded by Exon-17 can be detected by aso-called sandwich ELISA (Enzyme-linked immunosorbent assay). When adiagnostic kit is used, a sample is initially contacted with a plate onwhich a primary antibody has been immobilized to form a complex, and asecondary antibody labeled with a marker is bound to this complex, andthen the signal intensity of the marker in this ternary complex ismeasured, whereby a periostin isoform having a peptide region encoded byExon-17 can be detected or quantified. In particular, since a periostinisoform having a peptide region encoded by Exon-17 is a splice variantwhich is highly expressed during the pathology of cancer or the like andis involved in primary tumor growth and cancer metastasis, the pathologyin cancer or the like can be diagnosed by monitoring its production.

In this way, an antibody for use in the present invention can be usedhere as the secondary antibody by labeling of the antibody.

The following examples further illustrate the present invention indetail and specifically, without, however, thus limiting the invention.

EXAMPLES Preparation Example 1 Search for Periostin by Subtraction

1-1 Preparation of Pathologic Model Rats of Heart Failure and Collectionof Left Ventricular Samples

Male Dahl salt-sensitive rats (Dahl-S) (Shimizu Laboratory Supplies)were raised on an 8% high salt diet from 6 weeks of age, and the leftventricle was collected from three animals each at cardiac hypertrophystage (11 weeks of age) and heart failure stage (14 weeks of age).

1-2 Preparation of mRNA

Total RNA was prepared from about 500 mg of the left ventricle usingISOGEN (Nippon Gene) as instructed by the manufacturer. Then, mRNA waspurified from about 40 μg of the combined total RNA from three animalseach at cardiac hypertrophy stage and heart failure stage using FastTrack 2.0 Kit (Invitrogen) as instructed by the manufacturer to recoverabout 3 μg of mRNA at each stage.

1-3 cDNA Subtraction

cDNA subtraction was performed using PCR-Select cDNA subtraction kit(Clontech) as instructed by the manufacturer. That is, cDNA wassynthesized from 2 μg of each mRNA obtained in 1-2 above and digestedwith restriction enzyme RsaI. Then, subtraction hybridization wasperformed using the cDNA synthesized from the animals at 14 weeks of ageas tester cDNA and the cDNA synthesized from the animals at 11 weeks ofage as driver cDNA after 2 adapters included in the kit had beenseparately linked to the tester cDNA. Then, a cDNA fragment with alteredexpression level was specifically amplified by PCR using primerscomplementary to the adapters to give amplification product 1.

A similar subtraction operation was performed using the cDNA synthesizedfrom the animals at 11 weeks of age as tester cDNA and the cDNAsynthesized from the animals at 14 weeks of age as driver cDNA to giveamplification product 2.

1-4 Dot Blot Screening

A. Preparation of Dot Blots

Amplification product 1 was TA cloned into a PCR II vector (Invitrogen)and clones having the insert fragment were selected. The insert fragmentof each clone was amplified by PCR reaction, and then 1 μl each of thereaction solution was heat-treated and then dot-blotted on 2 nylonmembrane filters (Boehringer) and fixed with a UV crosslinker(Stratagene).

B. Preparation of cDNA Probes

Amplification product 1 was digested with restriction enzymes RsaI andEaeI, SmaI to remove the adapters and subjected to random prime labelingwith DIG-dUTP using DIG High Prime DNA labeling/detection kit II(Boehringer) as instructed by the manufacturer to prepare cDNA probe 1.Similarly, cDNA probe 2 was prepared from amplification product 2.

C. Screening

One of the dot blot membranes prepared in A above was hybridized withcDNA probe 1 and the other with cDNA probe 2. Specifically,hybridization was performed in DIG Easy Hyb solution at 42° C. overnightusing DIG High Prime DNA labeling/detection kit II (Boehringer) asinstructed by the manufacturer. The membranes were washed twice with2×SSC, 0.1% SDS at room temperature for 5 minutes and twice with0.1×SSC, 0.1% SDS at 68° C. for 15 minutes, and then reacted withalkaline phosphatase-labeled DIG antibodies in the blocking bufferincluded in the kit, and then CSPD ready-to use was added to advancechemiluminescence and X-ray film was exposed. Clones showing a strongersignal in cDNA probe 1 than cDNA probe 2 were selected as positiveclones and sequenced.

1-5 Sequencing

The nucleotide sequences were determined by analysis on an automatic DNAsequencer model 373A (PE Applied Biosystems) using THERMO Sequenase™ IIdye terminator cycle sequencing kit (Amersham Pharmacia). Thus obtainedgene sequences were compared with sequences in the GenBank databank toreveal that one of the clones (SF014) was a gene having an 86% homologyto mouse periostin (GenBank Accession No. D13664).

Preparation Example 2 Cloning of Rat Periostin cDNA

Rat periostin cDNA was isolated by screening 10 phage subpools of about4000 clones (a total of about 40,000 clones) prepared from a rat aortacDNA library (Clontech) inserted into λgt11 vector by PCR using primers(1) 5′-GTTCATTGAAGGTGGCGATGGTC-3′ (SEQ ID NO: 15), and (2)5′-GAGATAAAATCCCTGCATGGTCCT-3′ (SEQ ID NO: 16) designed on the basis ofthe nucleotide sequence of SF014 to give 3 positive subpools. One of thesubpools was screened by hybridization using the fragment amplified byPCR as a probe labeled with alkaline phosphatase using AlkPhos Direct™(Amersham Pharmacia) to give one positive clone rat periostin #1. Itsinsert fragment was subcloned into the EcoRI site of pBluescript II(Stratagene) and the total nucleotide sequence was determined accordingto the method of Preparation example 1-5.

The resulting clone had a length of about 3 kb corresponding tonucleotide 292 to the 3′ end of mouse periostin (GenBank Accession No.D13664), suggesting that it was a 5′-truncated clone.

Thus, SMART™ RACE cDNA Amplification Kit (Clontech) was used asinstructed by the manufacturer to perform 5′-RACE reaction using rataorta cDNA as a template and the primer (2)5′-GAGATAAAATCCCTGCATGGTCCT-3′ (SEQ ID NO: 16) and a primer (3)5′-CACGGTCGATGACATGGACAACACC-3′ (SEQ ID NO: 17) designed on the basis ofthe nucleotide sequence of rat periostin #1. The resulting PCR productwas TA cloned into PCR II vector of Invitrogen to give a clonedesignated as rat periostin 5′RACE #1. The nucleotide sequence wasdetermined according to the method of Preparation example 1-5.

The results showed that rat periostin 5′RACE #1 was a clone longer byabout 300 bp than the initially obtained rat periostin #1 in the 5′direction with the 5′ end being longer by 15 bp than the 5′ end of mouseperiostin (GenBank Accession No. D13664). Ten phage subpools of about40,000 clones (a total of about 400,000 clones) prepared from the rataorta cDNA library were screened by PCR using a primer (4)5′-ACGGAGCTCAGGGCTGAAGATG-3′ (SEQ ID NO: 18) designed on the basis ofthe nucleotide sequence of rat periostin 5′RACE #1 and the primer (3)5′-CACGGTCGATGACATGGACAACACC-3′ (SEQ ID NO: 17) to give 2 positivesubpools. One of the subpools was screened by hybridization using thefragment amplified by PCR as a probe to give one positive clonedesignated as rat periostin #2. The insert fragment was subcloned intothe EcoRI site of pBluescript II (Stratagene) and the nucleotidesequence was determined according to the method of Preparation example1-5.

The resulting clone had a length of about 2.6 kb with the 5′ end beingthe same as that of the clone obtained with 5′-RACE and the 3′ endcorresponding to up to nucleotide 2410 of mouse periostin (GenBankAccession No. D13664). The nucleotide sequence of rat periostin 5′RACE#1 previously obtained was exactly the same as the nucleotide sequenceof the relevant region of rat periostin #2. The full length of ratperiostin cDNA was completed by rat periostin #1 and rat periostin #2.The nucleotide sequence of this full-length cDNA and the amino acidsequence translated from this nucleotide sequence are shown as SEQ IDNOs: 6 and 1.

Preparation Example 3 Construction of a Myc-His-rat Periostin FusionProtein Expression Vector

An expression vector having a Myc epitope and 6 histidine tags at thecarboxyl terminus of the protein translated from the coding region ofthe rat periostin gene obtained in Preparation example 2, and having aCMV promoter, was prepared.

Initially, a fragment of about 500 bp obtained by digestion of ratperiostin 5′RACE #1 obtained in Preparation example 2 with restrictionenzymes EcoRI and HindIII and a fragment of about 2780 bp obtained bydigestion of rat periostin #1 obtained in Preparation example 2 withrestriction enzymes HindIII and HpaI were ligated to a vector fragmentobtained by digesting pTracer-CMV2 vector (Invitrogen) with restrictionenzymes EcoRI and EcoRV using a ligation kit (Takara Bio Inc.) to give aplasmid designated as pTracer-CMV2/rat periostin. Thus preparedpTracer-CMV2/rat periostin was digested with restriction enzymes EcoRIand SmaI to give a fragment of about 2330 bp containing the codingregion of the rat periostin gene, and PCR was performed using ratperiostin #1 obtained in Preparation example 2 as a template and primer(5) 5′-GACCCGGGAAGAACGCATCATC-3′ (SEQ ID NO: 19) designed on the basisof the sequence of the template and primer (6)5′-TGGGTGACCCTGAGAACGGCCTTCTCTTGATC-3′ (SEQ ID NO: 20) designed toinsert a BstEII site immediately before the stop codon of rat periostinand the amplification product was purified and then digested withrestriction enzymes SmaI and BstEII to give a fragment of about 270 bp.These two fragments were ligated to a vector fragment obtained bydigestion of an expression vector constructing plasmidpcDNA4/Myc-His/type C (Invitrogen) with restriction enzymes EcoRI andBstEII using a ligation kit (Takara Bio Inc.) to give a plasmiddesignated as pcDNA4/Myc-His/rat periostin. The total nucleotidesequence of the insert was confirmed by the method described inPreparation example 1-5.

Preparation Example 4 Construction of a Baculovirus Expression Vector

The plasmid pcDNA4/Myc-His/rat periostin obtained in Preparation example3 was digested with restriction enzymes SacI and PmeI to excise apeptide fragment rat PN-1/Myc-His. This fragment was ligated to a vectorfragment obtained by digesting pFastBacHTc (Invitrogen) with restrictionenzymes SacI and KpnI (blunting) using a ligation kit (Takara Bio Inc.)to give an expression vector designated as pFastBac/ratperiostin-1/Myc-His. The nucleotide sequence of the insert was confirmedby the method described in Preparation example 1-5.

Preparation Example 5 Preparation and Cultivation of a RecombinantBaculovirus

DH10BAC cells of Escherichia coli were transformed with pFastBac/ratperiostin-1/Myc-His obtained in Preparation example 5 to prepare arecombinant baculovirus. Electrophoresis and PCR confirmed that theresulting baculovirus contains the desired insert.

Insect Sf9 cells (2×10⁶ cells/mL) infected with this recombinantbaculovirus at MOI=0.1 were cultured in a serum-free medium (containing50 μg/mL gentamicin in 2000 mL of Sf-900IISFM (Invitrogen)) at 28° C.for 4-5 days and then the culture supernatant was harvested.

Preparation Example 6 Purification of Rat Periostin Protein

To an SP Sepharose Fast Flow column (10 mL bed volume) equilibrated withan equilibration buffer (50 mM sodium acetate buffer, pH 6.0, 0.1Msodium chloride) was applied 2000 mL of the culture supernatant obtainedin Preparation example 5, and the resulting flow-through fraction waspooled as an SP Sepharose flow-through fraction.

The column was washed with the equilibration buffer until the absorbanceat 280 nm approached 0 (about 100 mL) to give an SP Sepharose washfraction.

The column was eluted with 100 mL of an elution buffer (50 mM sodiumdihydrogen phosphate (pH8.0), 0.5M sodium chloride, 5 mM imidazole) togive an SP Sepharose eluate fraction.

Then, 100 mL of the SP Sepharose eluate fraction was applied to anNi-NTA agarose column (5 mL bed volume) equilibrated with 50 mM sodiumphosphate buffer, pH8.0, 0.5M sodium chloride and 5 mM imidazole, andthe resulting flow-through fraction was pooled as an Ni-NTA agaroseflow-through fraction.

The column was washed with about 50 mL of a washing buffer (50 mL sodiumdihydrogen phosphate, pH8.0, 0.5M sodium chloride, 5 mM imidazole) togive an Ni-NTA agarose wash fraction.

The column was eluted with about 25 mL each of elution buffers (1) 50 mMsodium dihydrogen phosphate, 0.5M sodium chloride, 20 mM imidazole,followed by similar compositions except that the imidazoleconcentrations were (2) 30 mM, (3) 40 mM, (4) 50 mM and (5) 60 mM togive Ni-NTA agarose eluate fractions (1)-(6).

Fractions shown to contain the desired protein by Western blotting wereconcentrated to 1 mL or less.

Then, the concentrated samples were applied to a gel filtration column(Sephacryl S-200HR φ11 mm×95 cm; 90 bed volume) equilibrated withdegassed PBS (−) (137 mM NaCl, 8.1 mM Na₂HPO₄, 2.68 mM KCl, 1.47 mMKH₂PO₄) and eluted with PBS (−) and the eluate was lyophilized to give apurified rat periostin protein.

Example 1 Synthesis of Rat Exon-17 Peptide Chain and Preparation ofPolyclonal Antibody Against it

A structure specific to rat PN-1 was identified as Exon-17 sequence bysequence comparison since heart dilation was induced by increasedexpression of the PN-1 gene in the heart of normal SD rats and thesurvival rate was improved by administration of an antisenseoligonucleotide against rat periostin to the heart of Dahl heart failuremodel rats and rat PN-1 was shown to have no cell adhesive properties incontrast to previously reported PN-2. A peptide having a Cys residueadded to the N-terminus of the amino acid sequence constituting thisExon-17 was chemically synthesized in 10 mg yield at a purity of 80% ormore. Rabbits (Kbl:JW) were immunized with the polypeptide coupled to 6mg of a carrier protein KLH. FCA (Freund's complete adjuvant) was usedin the primary immunization, and FIA (Freund's incomplete adjuvant) wasused in the secondary and subsequent immunizations. Administration wasperformed at 20 dorsal subcutaneous sites at weeks 0, 2, 4 and 6 using apeptide dose of 800 μg/animal in the primary immunization, and 400μg/animal in the secondary and subsequent immunizations. The antibodytiter was determined by ELISA and total sera were collected at week 7.Then, an affinity column was prepared by use of a synthetic peptide, andonly the antibody specifically reacting to the Exon-17 peptide wascollected. The polyclonal antibody against the peptide encoded byExon-17 of rat periostin is hereinafter referred to as anti-rat Exon-17peptide antibody.

Example 2 In Vitro Study of the Presence or Absence of Anti-CellAdhesive Activity of Rat Periostin Protein (Rat PN-1)

Rat heart fibroblasts were obtained by a method similar to thosedescribed in literature (Ruwhof C, van Wamel A E, Egas J M, van derLaarse A. Mol Cell Biochem. 2000 May; 208 (1-2):89-98, Ashizawa N, GrafK, Do Y S, Nunohiro T, Giachelli C M, Meehan W P, Than T L, Hsuch W A. JClin Invest. 1996 Nov. 15; 98 (10):2218-27). Specifically, 20 SD rats at1-2 days of age were anesthetized with ether and the chest wasdisinfected with ethanol. The heart was isolated and placed in a dishcontaining PBS (−), where the heart was transversely incised to bleed itand further washed with PBS (−) three times, and then PBS (−) wasdiscarded to the minimum possible level and the heart was minced withscissors. Then, the minced tissue was agitated in a 1:1 mixture of PBS(−): collagenase/trypsin at 37° C. for 15 minutes, and then cells werelysed by pipetting as thoroughly as possible. Then, the cell lysate wasfiltered through a platinum mesh in a centrifugal tube, and the platinummesh was washed with 10 ml of M199 medium containing 10% serum and 10 mlof PBS (−). Then, the centrifugal tube was spun at 1500 rpm for 10minutes, and the cells obtained as a pellet fraction were stirred in 20ml of M199 medium containing 10% serum and plated on a dish. The dishwas allowed to stand at 37° C. for 1 hour, and then cells adhered to thedish were collected as rat heart fibroblasts. The rat heart fibroblastswere plated on a 96-well plate at a density of 6.4×10⁴ cells/100 μl andcultured overnight, and then the culture was incubated in fresh DMEMmedium with 10% FBS containing 10 μg/ml cycloheximide at 37° C. for 1hour. Then, the cells were washed twice with DMEM medium (serum free)prewarmed at 37° C., and rat periostin protein (rat PN-1) preparedaccording to the Preparation examples was added to DMEM medium (serumfree) at a final concentration of 10 μg/ml. Fibronectin having celladhesion-promoting properties was used as a positive control and BSA(bovine serum albumin) was used as a negative control. After incubationat 37° C. for 1 hour, microscopy showed that all the cells wereseparated in the group treated with rat periostin protein (rat PN-1),and the cells were washed twice with PBS (−) and then fixed in 10%neutral buffered formalin for 30 minutes. Then, the cells were washedwith PBS (−) three times and then stained with crystal violet for 30minutes. Then, the degree of staining was measured using a plate readerat 550 nm (BIO-RAD, Model 680 MICRO PLATE READER) (FIG. 2). As a result,the control groups treated with fibronectin and BSA and the untreatedgroup did not show anti-cell adhesive properties because the adheredcells were not separated, in contrast to the group treated with ratperiostin protein (rat PN-1) in which the cells were separated, showingthat rat periostin protein (rat PN-1) has a separating effect on adheredcells, i.e., anti-cell adhesive properties.

Example 3 In Vitro Study of the Neutralizing Activity of Anti-RatExon-17 Peptide Antibody

SD rat heart fibroblasts obtained by a method similar to that of Example2 were plated on a 96-well plate at a density of 6.4×10⁴ cells/100 μland cultured overnight, and then the culture was incubated in fresh DMEMmedium with 10% FBS containing 10 μg/ml cycloheximide at 37° C. for 1hour. Then, the cells were washed twice with DMEM medium (serum free)prewarmed at 37° C., and rat periostin protein (rat PN-1) and anti-ratExon-17 peptide antibody were added to DMEM medium (serum free) at finalconcentrations of 10 μg/ml and 100 μg/ml, respectively. Rat periostinprotein alone was used as a positive control and BSA was used as anegative control. After incubation at 37° C. for 1 hour, microscopyshowed that all the cells were separated in the group treated with ratperiostin protein alone, and the cells were washed twice with PBS (−)and then fixed in 10% neutral buffered formalin for 30 minutes. Then,the cells were washed with PBS (−) three times and then stained withcrystal violet for 30 minutes. Then, the degree of staining was measuredusing a plate reader at 550 nm (BIO-RAD, Model 680 MICRO PLATE READER)(FIG. 3). The results showed that anti-rat Exon-17 peptide antibody isan antibody having the activity of inhibiting rat periostin protein(PN-1)-induced separation of adhered cells, i.e., inhibiting theanti-cell adhesive properties of PN-1, i.e., neutralizing the anti-celladhesive properties of rat periostin protein (rat PN-1).

Example 4 Effect of Anti-Rat Exon-17 Peptide Antibody on AcuteMyocardial Infarction Model Rats

A male Lewis rat weighing 250-300 g was fixed on a rat surgical tableafter the animal was thoroughly anesthetized by peritonealadministration of pentobarbital (0.1 ml/100 g). A tube was orallyinserted into the trachea and connected to a rat ventilator (tidalvolume 3 ml, 80 breaths/min), and the skin was laterally incised fromthe left third intercostal space of the sternum and the underlyinggreater pectoral muscle was also laterally incised, and the intercostalspace was opened using a rat rib spreader to expose the heart.

Then, the left coronary artery nearly beneath the left atrium wasligated with 1.0 silk using a curved needle having a diameter of 5 mm.After visual confirmation that the anterior and lateral walls alongwhich the left coronary artery runs had been changed from red to whiteto show sufficient blockage of the coronary bloodstream and thedisappearance of wall motion at these sites (in the sham operationgroup, the needle was passed through the coronary artery and thenremoved without ligation), the third and fourth ribs were fixed byligation with 3.0 silk (after the lung was expanded to remove the airexisting outside the lung in the rib cage so that the lung can be easilyexpanded). The incision site in the skin was sutured with 3.0 silk inthe same manner and then observed for a while, and the tube was removedafter confirmation of recovery of consciousness and resumption ofspontaneous breathing.

Acute myocardial infarction models were sequentially prepared by theforegoing procedure.

On the following day, percutaneous echocardiography was performed underintranasal anesthesia with isoflurane, and small infarction modelshaving an infarction size less than 20% of the entire periphery of theleft ventricle were excluded. The remaining infarction models wereranked in order of increasing heart function, and alternately classifiedinto a group treated with anti-rat Exon-17 peptide antibody and a grouptreated with a control antibody (rabbit IgG) each 200 μg via tail vein.

The antibodies were administered to each group on the day following thepreparation of the models and at intervals of 6 days after the initialadministration, a total of 4 times.

The heart was evaluated by echocardiography through the chest wall atintervals of one week until the end of 8 weeks. At the end of 8 weeks, atube was inserted into the trachea and connected to a ventilator underanesthesia with pentobarbital, and the skin was incised from the leftneck and the neck muscles were retracted with forceps to expose the leftcommon carotid artery, and after bleeding was stopped by ligation at theorigin of the left carotid artery, the artery was pierced at a distalsite by small scissors and a rat mirror catheter was inserted from thatsite in such a manner that the catheter tip with a pressure sensorreached the inside of the left ventricle while the catheter wasconnected to a computer to measure heart function and blood pressure andthe like.

The results of echocardiography 4 weeks after the preparation of themodels showed that the reduction of the anterior wall thickness andposterior wall thickness of the heart was inhibited, the increase of theend-diastolic inner diameter and end-systolic inner diameter wasinhibited, and that the EF value indicative of the contractile functionof the heart increased in the group treated with anti-rat Exon-17peptide antibody significantly as compared with the control grouptreated with rabbit IgG. In brief, heart dilation was inhibited, showingthat heart function was improved (FIG. 4-1 to FIG. 4-3). The results ofechocardiography 8 weeks after the preparation of the models also showedinhibition of heart dilation and improvement of heart function in thesame manner as the results after 4 weeks (FIG. 5-1 to FIG. 5-3). Theseresults suggested that the effect of anti-rat Exon-17 peptide antibodyinhibiting heart dilation and improving heart function is maintainedeven about 4 weeks after the administration of the antibody. Then,hemodynamics showed significant differences in maximum derivative ofleft ventricular pressure ((+)dP/dt), minimum derivative of leftventricular pressure ((−)dP/dt) and left ventricular end-diastolicpressure (LVEDP) in the group treated with anti-rat Exon-17 peptideantibody as compared with the control group treated with IgG, suggestingthat heart function was improved (FIG. 6-1 to FIG. 6-3). In heartsections stained with Masson trichrome, blue sites decreased in thegroup treated with anti-rat Exon-17 peptide antibody as compared withthe control group treated with IgG, showing that fibrosis was inhibited(FIG. 7). Analysis of the minor axis diameters of myocardial cellsshowed that the reduction of the minor axis diameters of myocardialcells was significantly inhibited in the group treated with anti-ratExon-17 peptide antibody as compared with the control group treated withIgG (FIG. 8). This result correlates with the result ofechocardiography. Moreover, the results of gene expression analysis ininfarct sites and non-infarct sites showed that the expression levels ofendothelin-1 (ET-1), collagen type I, III, and TGF-beta at non-infarctsites significantly decreased in the group treated with the neutralizingantibody as compared with the group treated with the control antibody tolevels comparable to those of the sham group, indicating that thecondition was improved (FIG. 9-1 to FIG. 9-5).

Example 5 Cloning of Full-Length Human Periostin-1 cDNA

cDNA prepared from 1 μg of human heart-derived total RNA (Clontech,catalog No, 64100-1, lot No. 4120493) was used as a template to performPCR with KOD plus DNA polymerase (Toyobo Co., Ltd.) using the followingprimers for full-length cloning: sense chain5′-AAGCTAGCCACCATGATTCCCTTTTTACCCAT-3′ (SEQ ID NO: 27) and antisensechain 5′-AACTCCACAATTTCCCTCAT-3′ (SEQ ID NO: 28). The resulting PCRproducts were cloned using a Zero Blunt TOPO PCR Cloning kit(Invitrogen).

A sense chain 5′-TAACCAAAGTTGTGGAACCAA-3′ (SEQ ID NO: 29) was preparedfrom a region corresponding to human periostin Exon-17, while anantisense chain 5′-TGTGTCTCCCTGAAGCAGTC-3′ (SEQ ID NO: 30) was preparedfrom a region corresponding to Exon-21, followed by selection of clonesdetected with these primers from this clone group. Then, the nucleotidesequences of the selected clones were determined to select an unsplicedclone, thereby completing the full-length cloning of human periostin-1cDNA. The resulting clone was designated as pCR4/human periostin-1.

Example 6 Construction of Human Periostin-1 Expression Vector for InVitro Translation

The plasmid pCR4/human periostin-1 obtained in Example 5 was digestedwith restriction enzymes Pme I and Not I to excise a DNA fragment humanperiostin-1, which was then blunted. A pTNT expression vector (Promega),into which CATCACCATCACCATCACTAA (6×His+termination codon) (SEQ ID NO:31) had been inserted, was enzymatically digested at the Mlu I site inits multicloning site and then blunted. The DNA fragment obtained abovewas ligated to this vector using a ligation kit (TaKaRa Bio Inc.).

Then, for the purpose of creating in-frame fusion with the His tag,synthetic linkers (sense chain5′-CTAGAAGACGATTAAGGGAAGGTCGTTCTCAGCTGGAAGTTCTGTTCCAGGGGCCC-3′ (SEQ IDNO: 32) and antisense chain5′-GGGCCCCTGGAACAGAACTTCCAGCTGAGAACGACCTTCCCTTAATCGTCTT-3′ (SEQ ID NO:33)) were prepared and ligated to the vector fragment digested withrestriction enzymes Xba I and Sma I using a ligation kit. The nucleotidesequence of the ligated part was confirmed, and the resulting expressionvector was designated as pTNT/human periostin-1/His.

Example 7 Protein Synthesis by In Vitro Translation

The expression vector obtained in Example 6 was provided for in vitroprotein synthesis with TNT SP6 Quick Coupled Transcription/TranslationSystems (Promega). More specifically, relative to 2 μg of the pTNT/humanperiostin-1/His expression vector, 40 μl of SP6 Quick Master Mix and 1μl of 1 mM methionine were added and diluted with DEPC-treated water togive a total volume of 50 μl, followed by reaction at 30° C. for 90minutes. The reaction product was stored at −80° C. until purification.

Example 8 Purification of Human Periostin Protein (PN-1)

The synthetic protein obtained in Example 7 was purified using a MagZProtein Purification. System (Promega). More specifically, to thesynthetic protein obtained in Example 7, 2 volumes of MagZ Binding/Washbuffer were added and mixed well. The sample thus prepared was added toMagZ Binding Particles. After stirring of this mixture at 4° C. for 1hour, the supernatant was removed and the MagZ Binding Particles werewashed four times with MagZ Binding/Wash buffer, followed by elution ofthe synthetic protein with MagZ Elution buffer. The protein thuspurified was stored at −80° C. until use.

Example 9 Preparation of Monoclonal Antibody Against Human PeriostinExon-17 Peptide Chain (1) Antigen Preparation

A peptide (SEQ ID NO: 25) having a Cys residue added to the N-terminusof the amino acid sequence constituting human periostin Exon-17 (SEQ IDNO: 4) was chemically synthesized by the Fmoc method to obtain thepeptide in 10 mg yield at a purity of 90% or more. As a carrier protein,KLH (5 mg, CALBIOCHEM) was coupled to this peptide to give an antigensolution. Namely, KLH was dissolved in PBS (0.01M) and adjusted to 3.3mg/mL, to which a 0.2524 mg/mL MBS solution (GE Healthcare Bio-SciencesKK) was then added dropwise and reacted with stirring at roomtemperature for 60 minutes. Dichloromethane was used to remove free MBS,to thereby obtain KLH-MB. This KLH-MB (5 mg) was mixed with the antigenpeptide (5 mg) dissolved in 0.01M sodium phosphate buffer (pH7.2) andreacted with stirring at 4° C. for 12 hours to obtain the antigensolution.

(2) Immunization

Three female BALB/c mice at 6 weeks of age were each subcutaneouslyinjected into both soles with the whole volume of a mixed emulsion ofthe antigen solution (50 μl) containing 100 μg KLH-coupled antigenpeptide obtained in (1) and FCA (Freund's complete adjuvant, 50 μl). Themice were then injected twice into both soles with an in situ preparedmixed emulsion of the above antigen solution and FIA (Freund'sincomplete adjuvant) at an interval of 2 weeks. The mice were thensacrificed by cervical dislocation and lymph nodes in their soles wereaseptically collected.

While supplying RPMI medium (Kohjinbio Co., Ltd.), the above lymph nodeswere crushed and passed through a mesh of about 10 μm pore size toobtain lymph node cells suspended in RPMI medium. This suspension wascentrifuged at 1000 rpm for 10 minutes to obtain lymph node cells as apellet fraction. After this pellet fraction was hemolyzed to remove redblood cells in a solution (1 ml) prepared by adding 20 mM HEPES buffer(pH7.4) to a 0.84% ammonium chloride solution, centrifugation wasrepeated at 1,000 rpm for 5 minutes. The resulting pellet fraction (cellfraction) was washed several times with RPMI medium and then used forcell fusion.

(3) Preparation of Myeloma Cells

The mouse myeloma cell line P3X63Ag8U.1 (P3U1) that was resistant to8-azaguanine and secreted no immunoglobulin was cultured in RPMI mediumcontaining 20% fetal calf serum (FCS) in a 10% CO₂, 37° C. incubator.Cells in the logarithmic growth phase were collected and centrifuged at1,000 rpm for 5 minutes to obtain the cells alone as a pellet fraction,which were then suspended in RPMI medium.

(4) Cell Fusion

The RPMI medium obtained in (2) containing 10⁸ to 3×10⁸ immunized lymphnode cells and the RPMI medium obtained in (3) containing 10⁸ myelomacells were mixed and then centrifuged at 1,000 rpm for 10 minutes. Thesupernatant was gently removed to obtain the cells as a pellet fraction,followed by addition of 1 ml of 25% (w/v) polyethylene glycol 1500 (PEG1500, Boehringer). The cells were further diluted to a total volume of10 ml by slow addition of RPMI medium. To this suspension, 20%FCS-containing RPMI medium (10 ml) was added and allowed to stand for awhile, followed by centrifugation at 1,000 rpm for 5 minutes. Theresulting pellet fraction (cell fraction) was adjusted to a cell densityof 10⁶ cells/ml by addition of 20% FCS-containing RPMI, and this cellsuspension was dispensed at 200 μl/well in 96-well culture plates(Corning). After culturing in a 5% CO₂, 37° C. incubator for 24 hours,HAT solution (Invitrogen) was added and culturing was continued for anadditional 2 weeks.

(5) Screening by ELISA

Screening was performed to determine positive wells showing a reactionbetween the culture supernatant and the antigen peptide.

For use as an antigen solution for assay, the antigen peptide (2 mg)obtained in (1) was coupled to ovalbumin (OVA) as a carrier protein toprepare a conjugate.

Each well of a 96-well microtiter plate (Falcon 353912) was coated withthe above conjugate (1 μg/ml) by standing overnight at 4° C. Afterwashing this plate, the culture supernatant from (4) (50 μl, containingmonoclonal antibodies) was added dropwise to each well and allowed tostand in a 37° C. incubator for 2 hours, followed by washing with PBS(−) (phosphate buffered saline). After addition of alkalinephosphatase-conjugated sheep anti-mouse IgG antibody (Zymed), the platewas allowed to stand in a 37° C. incubator for 1 hour, washed with PBS(−) and then color developed for 20 minutes by addition of a colordevelopment substrate (ALP). The absorbance (antibody titer) at OD 490nm was measured for each well with a plate reader (BIO-RAD, Model 680MICRO PLATE READER) to confirm its reactivity with the antigen peptide,to thereby determine positive wells showing a reaction between theculture supernatant and the antigen peptide.

(6) Cloning of Antibody-Producing Cells

Cells in the positive wells whose reactivity with the antigen peptidewas confirmed by ELISA in (5) were provided for cloning ofantibody-producing cell lines by limiting dilution. Namely, cells in thepositive wells were plated into each well of a 96-well culture plate andcultured in a 5% CO₂, 37° C. incubator for 2 weeks. In the same manneras used in (5), reactivity with the antigen peptide was confirmed byELISA for the culture supernatant in each well, and cloning by limitingdilution was repeated again for each positive well to obtain 30 cellshaving a high reactivity with the antigen peptide and showing goodcolony growth. These cells were transferred to 24-well culture platesand cultured in a 5% CO₂, 37° C. incubator for 2 weeks. In the samemanner as used in (5), reactivity with the antigen peptide (antibodytiter) was confirmed again by ELISA for each culture supernatant. Cellsin 10 wells showing a high absorbance at OD 490 nm, i.e., 10 hybridomacell lines were determined to be useful as antibody-producing cells andwere selected.

Hybridoma cell lines No. OD value 1 0.41 2 0.37 3 0.68 4 0.24 5 0.33 60.32 7 0.33 8 0.32 9 0.12 10 0.3

Since the antibody-producing cells thus obtained always produce theantibodies of the present invention, i.e., anti-human Exon-17 monoclonalantibodies, the supernatant of the culture in which theseantibody-producing cells were cultured can be directly used as theantibody solution of the present invention. It is to be noted that theabove antibody-producing cell line (hybridoma) No. 1 (SBM337), whichproduces anti-human Exon-17 monoclonal antibody, was deposited underFERM BP-10718 on Nov. 1, 2006 with the International Patent OrganismDepositary, the National Institute of Advanced Industrial Science andTechnology.

(7) Confirmation of Binding Capacity to Human Periostin Protein (PN-1)

Antibodies produced by the 10 antibody-producing cells obtained in (6)were confirmed for their binding capacity to human periostin protein(PN-1) by dot blotting. Namely, the synthetic protein obtained inExample 8 (30 μg/ml) was spotted in 5 μl volumes on a Hybond-ECLnitrocellulose membrane (GE Healthcare Bio-Sciences KK) and washed oncewith TBS solution (10 mM Tris-HCl (pH8.0), 150 mM NaCl). Blocking buffer(Block Ace, Snow Brand Milk Products Co., Ltd.) was added and shaken atroom temperature for 1 hour. After a 1 μg/ml solution of each monoclonalantibody (primary antibody) obtained in (6) was added to the membraneand shaken for 3 hours, the membrane was washed four times with TBSsolution under shaking for 10 minutes. After a 0.4 μg/ml solution of anHRP-labeled anti-mouse IgG antibody (Promega) (secondary antibody) wasadded to the membrane and shaken at room temperature for 1 hour, themembrane was washed four times with TBS solution under shaking for 10minutes. Detection reagents (ECL plus western blotting detection system,GE Healthcare Bio-Sciences KK) were added and reacted for 1 minute todetect chemiluminescence. As a result, it was confirmed that all of the10 antibody-producing cells cloned in (6) bind to human periostin PN-1.

(8) Mass Production and Purification of Monoclonal Antibody

BALB/c mice were intraperitoneally administered with pristane[2,6,10,14-tetramethyl pentadecane (0.5 ml, Wako Pure ChemicalIndustries, Ltd.) and kept for 2 to 3 weeks. The monoclonalantibody-producing hybridomas No. 1 and No. 3 which had been maintainedat the logarithmic growth phase were collected and centrifuged to removethe culture supernatant. To the cells in each pellet fraction, FCS-freeRPMI medium was added to prepare a cell suspension at a cell density of1×10⁷ cells/ml. This cell suspension was intraperitoneally injected intothe BALB/c mice pretreated with pristane and, after about three weeks,the exuded ascites fluid was collected from the abdominal region by asyringe. After each collected ascites fluid was filtered using a filterwith a pore size of φ0.22 μm, the filtrates were purified in a routinemanner by affinity chromatography on a Protein G-sepharose column(Millipore, 11511324) to prepare two anti-human Exon-17 monoclonalantibodies.

Example 10 Recognition Site Analysis of Anti-Human Exon-17 MonoclonalAntibody in Human Periostin Exon-17 Peptide Chain

The resulting two monoclonal antibodies (No. 1 and No. 3) were analyzedfor their recognition sites in the human periostin Exon-17 peptide chain(epitope identification). Namely, based on an amino acid sequenceconsisting of 45 amino acids in total between the −9th phenylalaninefrom the N-terminus and the 9th isoleucine from the C-terminus of thehuman periostin Exon-17 peptide chain (SEQ ID NO: 4; the 1st threonineup to the 27th glutamic acid), the following 36 peptides composed of 10amino acids were synthesized on a cellulose membrane to prepare amembrane-bound peptide array (custom SPOTs service of Sigma-AldrichJapan K.K.).

1 FKEIPVTVYT (SEQ ID NO: 35) 2 KEIPVTVYTT (SEQ ID NO: 36) 3 EIPVTVYTTK(SEQ ID NO: 37) 4 IPVTVYTTKI (SEQ ID NO: 38) 5 PVTVYTTKII(SEQ ID NO: 39) 6 VTVYTTKIIT (SEQ ID NO: 40) 7 TVYTTKIITK(SEQ ID NO: 41) 8 VYTTKIITKV (SEQ ID NO: 42) 9 YTTKIITKVV(SEQ ID NO: 43) 10 TTKIITKVVE (SEQ ID NO: 44) 11 TKIITKVVEP(SEQ ID NO: 45) 12 KIITKVVEPK (SEQ ID NO: 46) 13 IITKVVEPKI(SEQ ID NO: 47) 14 ITKVVEPKIK (SEQ ID NO: 48) 15 TKVVEPKIKV(SEQ ID NO: 49) 16 KVVEPKIKVI (SEQ ID NO: 50) 17 VVEPKIKVIE(SEQ ID NO: 51) 18 VEPKIKVIEG (SEQ ID NO: 52) 19 EPKIKVIEGS(SEQ ID NO: 53) 20 PKIKVIEGSL (SEQ ID NO: 54) 21 KIKVIEGSLQ(SEQ ID NO: 55) 22 IKVIEGSLQP (SEQ ID NO: 56) 23 KVIEGSLQPI(SEQ ID NO: 57) 24 VIEGSLQPII (SEQ ID NO: 58) 25 IEGSLQPIIK(SEQ ID NO: 59) 26 EGSLQPIIKT (SEQ ID NO: 60) 27 GSLQPIIKTE(SEQ ID NO: 61) 28 SLQPIIKTEG (SEQ ID NO: 62) 29 LQPIIKTEGP(SEQ ID NO: 63) 30 QPIIKTEGPT (SEQ ID NO: 64) 31 PIIKTEGPTL(SEQ ID NO: 65) 32 IIKTEGPTLT (SEQ ID NO: 66) 33 IKTEGPTLTK(SEQ ID NO: 67) 34 KTEGPTLTKV (SEQ ID NO: 68) 35 TEGPTLTKVK(SEQ ID NO: 69) 36 EGPTLTKVKI (SEQ ID NO: 70)

This membrane was allowed to stand in a small volume of methanol for 5minutes and was then washed three times with TBS solution. Blockingbuffer (casein, included in SPOTS) was added and stirred at roomtemperature for 2 hours. After a 1 μg/ml solution of each monoclonalantibody (primary antibody) obtained in Example 9(8) was added to themembrane and shaken for 3 hours, the membrane was washed three times inTBS solution under shaking for 10 minutes. After a 0.4 μg/ml solution ofan HRP-labeled anti-mouse IgG antibody (Promega) (secondary antibody)was added to the membrane and incubated for 2 hours, the membrane waswashed three times with TBS solution under shaking for 5 minutes.Detection reagents (SuperSignal West Pico, Pierce) were added andreacted for 1 minute to detect chemiluminescence. As a result,monoclonal antibodies No. 1 and No. 3 were found to react with and bindto only synthetic peptide No. 9 consisting of the amino acid sequenceYTTKIITKVV (SEQ ID NO: 26), i.e., a peptide consisting of an amino acidsequence covering from the −1st tyrosine to the 9th valine from theN-terminus of the amino acid sequence of the human periostin Exon-17peptide chain (SEQ ID NO: 4) or covering from the 669th tyrosine to the678th valine from the N-terminus of the amino acid sequence of humanperiostin PN-1 (SEQ ID NO: 2).

Example 11 Recognition Site Analysis of Anti-Rat Exon-17 PolyclonalAntibody in Human Periostin Exon-17 Peptide Chain

In the same manner as shown in Example 10, the polyclonal antibodyprepared in Example 1 was analyzed for its recognition site in the humanperiostin Exon-17 peptide chain (epitope identification). As a result,as in the case of the monoclonal antibodies in Example 10, thepolyclonal antibody was found to react with only synthetic peptide No.9, indicating that the polyclonal antibody specifically recognizes thesame site as the monoclonal antibodies. This suggests that antibodieshaving the same specificity are obtainable in both cases where a ratperiostin Exon-17 peptide is used as an antigen to prepare a polyclonalantibody and where a human periostin Exon-17 peptide is used as anantigen to prepare a monoclonal antibody.

Example 12 Confirmation of Binding Capacity to Rat Periostin Protein(PN-1)

The resulting two monoclonal antibodies (No. 1 and No. 3) were confirmedfor their binding capacity to rat periostin protein (PN-1) by dotblotting. Namely, the purified protein obtained in Preparation example 6(30 μg/ml) was spotted in 5 μl volumes on a Hybond-ECL nitrocellulosemembrane (GE Healthcare Bio-Sciences KK) and washed once with TBSsolution (10 mM Tris-HCl (pH8.0), 150 mM NaCl). Blocking buffer (BlockAce, Snow Brand Milk Products Co., Ltd.) was added and shaken at roomtemperature for 1 hour. After a 1 μg/ml solution of each monoclonalantibody (primary antibody) was added to the membrane and shaken for 3hours, the membrane was washed four times with TBS solution undershaking for 10 minutes. After a 0.4 μg/ml solution of an HRP-labeledanti-mouse IgG antibody (Promega) (secondary antibody) was added to themembrane and shaken at room temperature for 1 hour, the membrane waswashed four times with TBS solution under shaking for 10 minutes.Detection reagents (ECL plus western blotting detection system, GEHealthcare Bio-Sciences KK) were added and reacted for 1 minute todetect chemiluminescence. As a result, it was confirmed that theresulting two monoclonal antibodies also bind to rat periostin PN-1.

Example 13 Epitope Analysis of Anti-Human Exon-17 Monoclonal Antibody

The results of Example 10 indicated that the epitope site of eachanti-human Exon-17 monoclonal antibody recognizes an amino acid sequence(TTKIITKVV; SEQ ID NO: 22) covering from the N-terminal threonine to the9th valine of the human periostin Exon-17 peptide chain (SEQ ID NO: 4).Likewise, the results of Example 12 confirmed that each anti-humanExon-17 monoclonal antibody also binds to rat periostin protein (PN-1).These results suggested that the epitope site of each anti-human Exon-17monoclonal antibody recognizes a region, whose amino acids do not differbetween humans and rats, in the amino acid sequence (TTKIITKVV; SEQ IDNO: 22) covering from the N-terminal threonine to the 9th valine of thehuman periostin Exon-17 peptide chain (SEQ ID NO: 4), i.e., the entireamino acid sequence, or a part thereof, which covers from the N-terminalthreonine to the 7th lysine of the human periostin Exon-17 peptide chain(SEQ ID NO: 4) or the rat periostin Exon-17 peptide chain (SEQ ID NO:3). Thus, for further analysis of the epitope site, alanine scanning wasperformed.

Based on an amino acid sequence (YTTKIITKVV; SEQ ID NO: 26) coveringfrom the −1st tyrosine to the 9th valine from the N-terminus of thehuman periostin Exon-17 peptide chain (SEQ ID NO: 4), the following 10peptides modified to replace some amino acids by alanines weresynthesized at a purity of 80% or more.

 #1 YTTKIITKVV (SEQ ID NO: 71)  #2 ATTKIITKAA (SEQ ID NO: 72)  #3AATKIITKAA (SEQ ID NO: 73)  #4 AAAKIITKAA (SEQ ID NO: 74)  #5 AAAAIITKAA(SEQ ID NO: 75)  #6 AAAAAITKAA (SEQ ID NO: 76)  #7 ATTKIITAAA(SEQ ID NO: 77)  #8 ATTKIIAAAA (SEQ ID NO: 78)  #9 ATTKIAAAAA(SEQ ID NO: 79) #10 ATTKAAAAAA (SEQ ID NO: 80)

The synthetic peptides (1 mg each) were solubilized with 50 μl PBS (−),spotted in 1.5 μl volumes on a Hybond-ECL nitrocellulose membrane (GEHealthcare Bio-Sciences KK) and washed once with TBS solution. Blockingbuffer (Block Ace, Snow Brand Milk Products Co., Ltd.) was added andshaken at room temperature for 1 hour. After a 1 μg/ml solution of eachmonoclonal antibody (primary antibody) obtained in Example 9(8) wasadded to the membrane and shaken for 3 hours, the membrane was washedfour times with TBS solution under shaking for 10 minutes. After a 0.4μg/ml solution of an HRP-labeled anti-mouse IgG antibody (Promega)(secondary antibody) was added to the membrane and shaken at roomtemperature for 1 hour, the membrane was washed four times with TBSsolution under shaking for 10 minutes. Detection reagents (SuperSignalWest Pico, Pierce) were added and reacted for 1 minute to detectchemiluminescence.

As a result, the monoclonal antibodies were found to strongly react withsynthetic peptide #7 shown above (a peptide comprising alaninesubstitutions at the 1st and the 8-10th amino acids from the N-terminusof a peptide consisting of an amino acid sequence (YTTKIITKVV; SEQ IDNO: 26) covering from the −1st tyrosine to the 9th valine from theN-terminus of the human periostin Exon-17 peptide chain (SEQ ID NO: 4)),weakly react with synthetic peptides #1 (a peptide consisting of anamino acid sequence (YTTKIITKVV; SEQ ID NO: 26) covering from the −1sttyrosine to the 9th valine from the N-terminus of the human periostinExon-17 peptide chain (SEQ ID NO: 4)) and #2 (a peptide comprisingalanine substitutions at the 1st and the 9-10th amino acids from theN-terminus of a peptide consisting of an amino acid sequence(YTTKIITKVV; SEQ ID NO: 26) covering from the −1st tyrosine to the 9thvaline from the N-terminus of the human periostin Exon-17 peptide chain(SEQ ID NO: 4)), and more weakly react with synthetic peptides #3 (apeptide comprising alanine substitutions at the 1st, the 2nd and the9-10th amino acids from the N-terminus of a peptide consisting of anamino acid sequence (YTTKIITKVV; SEQ ID NO: 26) covering from the −1sttyrosine to the 9th valine from the N-terminus of the human periostinExon-17 peptide chain (SEQ ID NO: 4)) and #8 (a peptide comprisingalanine substitutions at the 1st and the 7-10th amino acids from theN-terminus of a peptide consisting of an amino acid sequence(YTTKIITKVV; SEQ ID NO: 26) covering from the −1st tyrosine to the 9thvaline from the N-terminus of the human periostin Exon-17 peptide chain(SEQ ID NO: 4)).

Example 14 Recognition Site Analysis of Anti-Rat Exon-17 polyclonalantibody in human periostin Exon-17 Peptide Chain

In the same manner as shown in Example 13, the polyclonal antibodyprepared in Example 1 was analyzed for its recognition site in the humanperiostin Exon-17 peptide chain (epitope identification). As a result,as in the case of the monoclonal antibodies in Example 13, thepolyclonal antibody was found to strongly react with synthetic peptide#7, weakly react with synthetic peptides #1 and #2, and still moreweakly react with synthetic peptides #3 and #8, indicating that thepolyclonal antibody specifically recognizes the same site as themonoclonal antibodies. This suggests that antibodies having the samespecificity are obtainable in both cases where a rat periostin Exon-17peptide is used as an antigen to prepare a polyclonal antibody and wherea human periostin Exon-17 peptide is used as an antigen to prepare amonoclonal antibody.

Example 15 In Vitro Study of the Presence or Absence of Anti-CellAdhesive Activity of Human Periostin Protein (Human PN-1)

In the same manner as shown in Example 2, human heart fibroblasts(Dainippon Pharmaceutical Co., Ltd., catalog No. CS-ABI-5118) wereplated on a 96-well plate at a density of 6.4×10⁴ cells/100 μl andcultured overnight, and then the culture was incubated in fresh CSCmedium (Cell System Corporation) with 10% FBS containing 10 μg/mlcycloheximide at 37° C. for 1 hour. Then, the cells were washed twicewith CSC medium (serum free) prewarmed at 37° C., and human periostinprotein (human PN-1) prepared according to the Examples was added to CSCmedium (serum free) at a final concentration of 1 μg/ml. Fibronectinhaving cell adhesion-promoting properties was used as a positive controland BSA (bovine serum albumin) having no cell adhesive properties wasused as a negative control. After incubation at 37° C. for 3.5 hours,microscopy showed that all the cells were separated in the group treatedwith human periostin protein, and the cells were washed twice with PBS(−) and then fixed in 10% neutral buffered formalin for 30 minutes.Then, the cells were washed with PBS (−) three times and then stainedwith crystal violet for 30 minutes. Then, the degree of staining wasmeasured using a plate reader at 550 nm (BIO-RAD, Model 680 MICRO PLATEREADER) (FIG. 10). As a result, the control groups treated withfibronectin and BSA and the untreated group did not show anti-celladhesive properties, in contrast to the group treated with humanperiostin protein (human PN-1) in which the cells were separated,showing that human periostin protein (human PN-1) has anti-cell adhesiveproperties.

Example 16 In Vitro Study of the Neutralizing Activity of Anti-HumanExon-17 Monoclonal Antibody

In the same manner as shown in. Example 3, human heart fibroblasts wereplated on a 96-well plate at a density of 6.4×10⁴ cells/100 μl andcultured overnight, and then the culture was incubated in fresh CSCmedium with 10% FBS containing 10 μg/ml cycloheximide at 37° C. for 1hour. Then, the cells were washed twice with CSC medium (serum free)prewarmed at 37° C., and human periostin protein (human PN-1) andanti-human Exon-17 monoclonal antibody (No. 1 or No. 3) were added toCSC medium (serum free) at final concentrations of 1 μg/ml and 200μg/ml, respectively. Human periostin protein (human PN-1) alone was usedas a positive control and BSA was used as a negative control. Afterincubation at 37° C. for 3.5 hours, microscopy showed that all the cellswere separated in the group treated with human periostin protein (humanPN-1) alone, and the cells were washed twice with PBS (−) and then fixedin 10% neutral buffered formalin for 30 minutes. Then, the cells werewashed with PBS (−) three times and then stained with crystal violet for30 minutes. Then, the degree of staining was measured using a platereader at 550 nm (BIO-RAD, Model 680 MICRO PLATE READER) (FIG. 11). Theresults showed that anti-human Exon-17 monoclonal antibodies areantibodies having the activity of inhibiting the anti-cell adhesiveproperties of human periostin protein (human PN-1), i.e., neutralizingthe anti-cell adhesive properties of human periostin protein (humanPN-1).

As shown above, the anti-cell adhesive properties of human periostinprotein (human PN-1) were inhibited by antibodies against Exon-17 ofhuman periostin protein (human PN-1) which specifically recognize asequence or a part thereof consisting of the N-terminal 1st to 6th aminoacids of Exon-17, suggesting that Exon-17, at least a peptide segment ora part thereof consisting of the N-terminal 1st to 6th amino acids ofExon-17 constitutes a region related to the anti-cell adhesiveproperties of human periostin protein (human PN-1).

Example 17 Effect of Anti-Human Exon-17 Monoclonal Antibody on AcuteMyocardial Infarction Model Rats

In the same manner as shown in Example 4, a male Lewis rat weighing250-300 g was fixed on a rat surgical table after the animal wasthoroughly anesthetized by peritoneal administration of pentobarbital(0.1 ml/100 g). A tube was orally inserted into the trachea andconnected to a rat ventilator (tidal volume 3 ml, 80 breaths/min), andthe skin was laterally incised from the left third intercostal space ofthe sternum and the underlying greater pectoral muscle was alsolaterally incised, and the intercostal space was opened using a rat ribspreader to expose the heart.

Then, the left coronary artery nearly beneath the left atrium wasligated with 1.0 silk using a curved needle having a diameter of 5 mm.After visual confirmation that the anterior and lateral walls alongwhich the left coronary artery runs have been changed from red to whiteto show sufficient blockage of the coronary bloodstream and thedisappearance of wall motion at these sites (in the sham operationgroup, the needle was passed through the coronary artery and thenremoved without ligation), the third and fourth ribs were fixed byligation with 3.0 silk (after the lung was expanded to remove the airexisting outside the lung in the rib cage so that the lung can be easilyexpanded). The incision site in the skin was sutured with 3.0 silk inthe same manner and then observed for a while, and the tube was removedafter confirmation of recovery of consciousness and resumption ofspontaneous breathing.

Acute myocardial infarction models were sequentially prepared by theforegoing procedure.

On the following day, percutaneous echocardiography was performed underintranasal anesthesia with isoflurane, and small infarction modelshaving an infarction size less than 20% of the entire periphery of theleft ventricle were excluded. The remaining infarction models wereranked in order of increasing heart function, and alternately classifiedinto a group treated with anti-human Exon-17 monoclonal antibody (No. 3)and a group treated with a control antibody (rabbit IgG) each 200 μg viatail vein.

The antibodies were administered to each group on the day following thepreparation of the models and at intervals of 6 days after the initialadministration, a total of 4 times.

The heart was evaluated by echocardiography through the chest wall atintervals of one week until the end of 4 weeks. The results ofechocardiography 4 weeks after the preparation of the models showed thatthe reduction of the anterior wall thickness and posterior wallthickness of the heart was inhibited, the increase of the end-diastolicinner diameter and end-systolic inner diameter was inhibited, and thatthe FS value or EF value indicative of the contractile function of theheart increased in the group treated with anti-human Exon-17 monoclonalantibody significantly as compared with the control group treated withrabbit IgG. In brief, heart dilation was inhibited, showing that heartfunction was improved (FIG. 12-1 to FIG. 12-3).

As shown above, in acute myocardial infarction model rats, effects ofinhibiting heart dilation and improving heart function were caused byantibodies against Exon-17 of human periostin protein (human PN-1) whichhave an epitope composed of at least a sequence consisting of theN-terminal 1st to 6th amino acids of Exon-17, suggesting that Exon-17 ofhuman periostin protein (human PN-1), especially a region comprising atleast a peptide segment consisting of the N-terminal 1st to 6th aminoacids of Exon-17 is a region related to heart dilation and reduced heartfunction following myocardial infarction.

Example 18 In Vitro Study of the Presence or Absence of Non-CellAdhesive Activity of Rat PN-1 Protein

To a 96-well cell culture multiwell plate, 10 μg/ml fibronectin, 100μg/ml BSA or 10 μg/ml PN-1 protein was added and coated overnight at 4°C. After removal of the protein solution from the wells, mouse melanomaB16-F10 cells (ATCC No. CRL-6475) suspended in DMEM (10% BSA,PC:penicillin, SM:streptomycin) were added at 10⁴ cells/well andcultured in a 37° C. incubator for 3 hours. The level of cell adhesionwas measured as follows. After removal of the culture supernatant, thecells were fixed in 2.5% glutaraldehyde for 30 minutes, stained with0.02% crystal violet and then measured for their absorbance at OD 550 nmwith a plate reader (BIO-RAD, Model 680 MICRO PLATE READER). Untreatedwells were stained as background samples and used to correct theabsorbance values for comparison purposes. Data analysis was made by theFisher's PLSD test (FIG. 13). As a result, the positive controlfibronectin showed cell adhesion, whereas the negative control BSAshowed no cell adhesion. The group treated with rat PN-1 protein showedno cell adhesion, indicating that rat PN-1 protein has the effect ofpreventing cells from adhering, i.e., non-cell adhesive properties.

Example 19 In Vitro Study of the Presence or Absence of Anti-CellAdhesive Activity of Rat PN-1 Protein

To a 96-well cell culture multiwell plate, mouse melanoma B16-F10 cellssuspended in DMEM (10% BSA, PC:penicillin, SM:streptomycin) were addedat 10⁴ cells/well and cultured overnight in a 37° C. incubator. Afterremoval of the culture supernatant, 10 μg/ml fibronectin (SIGMA), 100μg/ml BSA (SIGMA) or PN-1 protein was added and cultured in a 37° C.incubator for 1 to 3 hours. The level of cell adhesion was measured asfollows. After removal of the culture supernatant, the cells were fixedin 2.5% glutaraldehyde for 30 minutes, stained with 0.02% crystal violetand then measured for their absorbance at OD 550 nm with a plate reader(BIO-RAD, Model 680 MICRO PLATE READER). Untreated wells were stained asbackground samples and used to correct the absorbance values forcomparison purposes. Data analysis was made by the Fisher's PLSD test(FIG. 14). The cells were photographed with a Nikon COOLPIX4500 attachedto a stereoscopic microscope LEICA MZ16. As a result, the control groupstreated with fibronectin and BSA and the untreated group did not showanti-cell adhesive properties because the adhered cells were notseparated, in contrast to the group treated with rat PN-1 protein inwhich the cells were separated, indicating that rat PN-1 protein has aseparating effect on adhered cells, i.e., anti-cell adhesive properties.

Example 20 In Vitro Study of the Neutralizing Activity of Anti-RatExon-17 Polyclonal Antibody

In the same manner as shown in Example 19, to a 96-well cell culturemultiwell plate, mouse melanoma B16-F10 cells suspended in DMEM (10%BSA, PC:penicillin, SM:streptomycin) were added at 10⁴ cells/well andcultured overnight in a 37° C. incubator. After removal of the culturesupernatant, the culture was incubated in fresh DMEM medium with 10% FBScontaining 10 μg/ml cycloheximide at 37° C. for 1 hour. Then, the cellswere washed twice with DMEM medium (serum free) prewarmed at 37° C., andrat periostin protein and anti-rat Exon-17 polyclonal antibody wereadded to DMEM medium (serum free) at final concentrations of 10 μg/mland 100 μg/ml, respectively. Rat periostin protein alone was used as apositive control and BSA was used as a negative control. Afterincubation at 37° C. for 1 hour, microscopy showed that almost all thecells were separated in the group treated with rat periostin proteinalone, and the cells were washed twice with PBS (−) and then fixed in10% neutral buffered formalin for 30 minutes. Then, the cells werewashed with PBS (−) three times and then stained with crystal violet for30 minutes. Then, the degree of staining was measured using a platereader at 550 nm (BIO-RAD, Model 680 MICRO PLATE READER) (FIG. 15). Theresults showed that the anti-rat Exon-17 polyclonal antibody is anantibody having the activity of inhibiting PN-1 protein-inducedseparation of adhered cells, i.e., inhibiting the anti-cell adhesiveproperties of PN-1 protein, i.e., neutralizing the anti-cell adhesiveproperties of rat PN-1 protein.

Example 21 In Vitro Study of the Effect of Anti-Rat Exon-17 PolyclonalAntibody on Cell Proliferation

In the same manner as shown in Example 19, to a 96-well cell culturemultiwell plate, mouse melanoma B16-F10 cells suspended in DMEM (serumfree, PC:penicillin, SM:streptomycin) were added at 10⁴ cells/well andcultured overnight in a 37° C. incubator. After removal of the culturesupernatant, DMEM (serum free, PC:penicillin, SM:streptomycin) mediumsupplemented with anti-rat Exon-17 polyclonal antibody and rabbit IgGantibody, each at a final concentration of 100 μg/ml, 10 μg/ml or 1μg/ml, was added to the cells, and overnight culturing was repeatedagain. On the following day, the medium in each well was replaced byDMEM (10% BSA, PC:penicillin, SM:streptomycin), and a Cell Titer 96AQueous One Solution Cell Proliferation Assay kit (Promega) was used toadd 20 μl Cell Titer solution per 100 μl medium, followed by incubationat 37° C. for 1 hour. Then, the degree of staining was measured using aplate reader at 490 nm (BIO-RAD, Model 680 MICRO PLATE READER) (FIG.16A). Similary, in case of mouse 4T1 breast cancer cells, anti-ratExon-17 polyclonal antibody and rabbit IgG antibody, each at a finalconcentration of 200 μg/ml, 100 μg/ml or 50 μg/ml, was added to thecells, and then measured (FIG. 16B). The results showed that theanti-rat Exon-17 polyclonal antibody is an antibody having the activityof inhibiting cell proliferation at high concentration.

Example 22 Periostin Expression in Primary Tumor Using Model Mice forLung Metastasis of Mouse Melanoma B16-F10 Cells

At 2 weeks after injection of mouse melanoma B16-F10 cells, mouseprimary tumores were collected and the homogenized tissue was allowed tostand on ice for 15 minutes after addition of RIPA buffer (RIPA LysesBuffer 10×; Upstate), 180 mM Na₃VO₄, protease inhibitor cocktail(Nacalai Tesque, Inc.) and 200 mM NaF. The homogenate was thencentrifuged at 15000 rpm, 4° C. for 20 minutes to collect thesupernatant. The extracted proteins were measured for theirconcentration using DC protein assay reagents (BIO-RAD). Then, theproteins were mixed into 2× sample buffer (Laemmli Sample Buffer(BIO-RAD), 5% 2-mercaptoethanol) and treated by heating at 98° C. for 5minutes. The prepared protein sample was electrophoresed at 10 mA for180 minutes using Multigel II mini 7.5 (Daiichi Pure Chemicals Co.,Ltd.) and then transferred overnight onto an Immobilon-P TransferMembrane (MILLIPORE) at 30 V and 4° C. Then, the membrane was soaked in5% skimmed milk in PBS-T for 1 hour or in Blocking One-P (NacalaiTesque, Inc.) for 20 minutes and shaken at room temperature forblocking. Then, a primary antibody (anti-human Exon-17 monoclonalantibody (No. 3) at 1:500 dilution; anti-avian α-tubulin monoclonal IgGantibody (Sigma) at 1:5000 dilution as a control) was added and reactedovernight at 4° C., followed by washing three times in PBS-T for 5minutes. Then, a secondary antibody (Anti mouse IgG HRP (PROMEGA) at1:10000 dilution) was added and reacted for 1 hour at room temperature.The membrane was washed in PBS-T once for 10 minutes and then twice for30 minutes, followed by chemiluminescence band detection using anECL-Plus (Amersham Biosciences). The results indicated that normal lowerlimbs showed no periostin expression, whereas the tumor tissue showedenhanced expression (FIG. 17).

Example 23 Effect of Anti-Rat Exon-17 Polyclonal Antibody and Anti-HumanExon-17 Monoclonal Antibody Using Model Mice for Lung Metastasis ofMouse Melanoma B16-F10 Cells

Mouse melanoma B16-F10 cells were cultured in a 37° C. incubator, washedwith PBS and then treated with trypsin/EDTA to float and collect thecells. Then, the cells collected by centrifugation at 1500 rpm for 3minutes were counted to give 5×10⁵ cells/animal and suspended in 100 μlPBS. The adjusted cells were injected into the sole of C57BL/6N malemice (8 weeks of age) using an insulin syringe equipped with a 29GMyjector injection needle (TERMO). First of all, to study the effect ofthe two monoclonal antibodies (No. 1 and No. 3) obtained in Example 6,these antibodies (2 μg/animal) were each administered to the mice viathe jugular vein through an insulin syringe equipped with a 29G Myjectorinjection needle, simultaneously with the inoculation of the cells. As acontrol, Normal Rabbit IgG (R&D Systems) was used. At one week afteradministration of the cells and antibodies, the diameter of swellinglesions in the lower limbs was determined with a caliper to evaluate %increase in primary tumor size. As a result, the % increase in primarytumor size was 74.5±6.3% (n=11) in the control group, 17.8±4.4% (n=10)in the antibody (No. 1) group and 19.5±9.9% (n=10) in the antibody (No.3) group, indicating that both antibodies have the same inhibitoryactivity on cancer growth (FIG. 18).

Next, subsequent to the inoculation of mouse melanoma B16-F10 cells,antibody administration was started after a given period of time tostudy whether the antibody has an effect under conditions much closer toclinical cases. At one week after cell injection for the experimentusing anti-rat Exon-17 polyclonal antibody (0.75 mg/ml) and at 3 and 7days after cell injection for the experiment using anti-human Exon-17monoclonal antibody (No. 3) (2.14 mg/ml), each antibody (20 μg/animal)was administered to the mice via the jugular vein through an insulinsyringe equipped with a 29G Myjector injection needle. As a control,Normal Rabbit IgG (R&D Systems) was used. After cell injection, thediameter of swelling lesions in the lower limbs was determined weeklywith a caliper to evaluate % increase in primary tumor size. To avoidovergrowth of primary tumors which causes direct cancer invasion intothe subperitoneal space and leads to death of the mice, lower limbs withprimary tumors were excised at 2 weeks after cell injection. At 5 weeksafter cell injection, autopsy was performed to determine the presence orabsence of metastasis from primary tumor to lung and the number ofcolonies metastasized to lung. The increase in primary tumor size wasexpressed as a percentage of increment, assuming that the limb diameterbefore injection of mouse melanoma B16-F10 cells was set to 100, anddata analysis was made by the Student's t-test. Likewise, the metastasisrate from primary tumor to lung was analyzed by the χ² test, while thenumber of colonies metastasized to lung was analyzed by the Mann-Whitneytest.

In the experiment where anti-rat Exon-17 polyclonal antibody wasadministered, the % increase in primary tumor size at 2 weeks after cellinjection (in comparison with the limb diameter before cell injection)was 198.1±10.428% (n=10) in the control group and 163.2±21.015% (n=11)in the neutralizing antibody group (FIG. 19). On the other hand, themetastasis rate from primary tumor to lung was 57.1% (n=7) in thecontrol group and 0% (n=6) in the neutralizing antibody group,indicating that the metastasis was significantly inhibited in theneutralizing antibody group (FIG. 19). The number of coloniesmetastasized from primary tumor to lung was 1.143±0.553 in the controlgroup (FIG. 19).

Next, in the experiment where anti-human Exon-17 monoclonal antibody(No. 3) was administered, the % increase in primary tumor size at 1 weekafter cell injection (in comparison with the limb diameter before cellinjection) was 77.6±9.484% (n=12) in the control group and 48.9±7.060%(n=11) in the neutralizing antibody group, indicating that primary tumorgrowth was significantly inhibited in the neutralizing antibody group(P<0.05) (FIG. 20). Likewise, the metastasis rate from primary tumor tolung was 75% (n=12) in the control group and 40% in the neutralizingantibody group (n=10), indicating that the metastasis rate tended todecrease in the neutralizing antibody group (P=0.0964) (FIG. 20). Thenumber of colonies metastasized from primary tumor to lung was 9.41±4.38in the control group and 0.7±0.335 in the neutralizing antibody group,indicating that the number of metastasized colonies was significantlyinhibited in the neutralizing antibody group (P<0.05) (FIG. 20). Theseresults showed that the anti-rat Exon-17 polyclonal antibody has aninhibitory effect on lung metastasis of melanoma cells. It was furtherfound that the anti-human Exon-17 monoclonal antibody (No. 3) also hasan inhibitory effect on primary tumor growth.

Example 24 Effect of Anti-Rat Exon-17 Polyclonal Antibody Using ModelMice for Lung Metastasis of Mouse 4T1 Breast Cancer Cells

Mouse 4T1 cells (ATCC No. CRL-2539) were seeded in 10 cm Tissue CultureDishes (Greiner) using RPMI1640 (Gibco) containing 10% bovine serumalbumin (FBS) (Bio west) and a Penicillin-streptomycin Mixed solution(Nacalai Tesque, Inc.), and then cultured in a 37° C. incubator for 24hours. After removal of the culture supernatant, the cells were washedwith PBS and then allowed to float by treatment with trypsin/EDTA. Thecells were collected and centrifuged at 1500 rpm for 3 minutes, 1.5×10⁵of which were then subcultured in a 37° C. incubator for 72 hours. Cellsin the logarithmic growth phase were counted to give 1×10⁶ cells/animaland suspended in 100 μl PBS. The adjusted cells were injected into thesole of BALB/c female mice (8 weeks of age) using an insulin syringeequipped with a 29G Myjector injection needle (TERMO). An antibody (20μg/animal) was also administered to the mice via the jugular veinthrough an insulin syringe equipped with a 29G Myjector injectionneedle. In the experiment using anti-rat Exon-17 polyclonal antibody (1mg/ml), the antibody was administered simultaneously with cellinjection, and further administered at 1 and 2 weeks after cellinjection. As a control, Normal Rabbit IgG (R&D Systems) was used. Aftercell injection, the mice were weekly measured for their body weight andthe diameter of swelling lesions in their lower limbs with a caliper toevaluate primary tumor volume. Evaluation was accomplished as describedin Dethlefsen L A, et al. J. Natl. Cancer Inst., 40, 389 (1968) todetermine the volume according to the following equation: (solelength)×(square of sole width)/2. At 3 weeks after cell injection,autopsy was performed to determine body weight, the presence or absenceof metastasis from primary tumor to lung, and the number of coloniesmetastasized to lung. Data analysis was made by the Student's t-test foreach case. As a result, with respect to changes in body weight, therewas no significant difference between both groups until 2 weeks aftercell injection. After 3 weeks, however, the body weight was 17.50±0.703g (n=6) in the control group and 21.24±0.517 g (n=6) in the neutralizingantibody group, indicating that primary tumor growth was significantlyinhibited in the neutralizing antibody group (P<0.05) (FIG. 21).Moreover, the tumor volume of primary tumor at 1 week after cellinjection was 301.3±11.49 mm³ (n=10) in the control group and235.9±7.842 mm³ (n=10) in the neutralizing antibody group, indicatingthat primary tumor growth was significantly inhibited in theneutralizing antibody group (P<0.05). Likewise, the tumor volume ofprimary tumor at 2 weeks after cell injection was 842.4±34.71 mm³ (n=10)in the control group and 613.9±45.17 mm³ (n=10) in the neutralizingantibody group, indicating that primary tumor growth was significantlyinhibited in the neutralizing antibody group (P<0.05) (FIG. 22). At 3weeks after cell injection, the control group showed loss of limbs dueto necrosis, whereas the neutralizing antibody group showed no loss oflimbs. Further, evaluation of bone destruction caused by bone invasionof breast cancer cells from primary tumor was accomplished by measuringbone area and the number of osteoclast after HE staining and TRAPstaining was carried out to ancle bone of lower limb. The remaining bonearea after direct invasion to bone was 326656±53628.7 (n=5) in thecontrol group and 545756.8±65928.8 (n=5) in the neutralizing antibodygroup, indication that bone significantly remained in the neutralizingantibody group (P<0.05) (FIG. 23). Moreover, the number of osteoclastwas adversely correlated the remaining bone area, and the number ofosteoclast was 49±9.576 (n=5) in the control group and 20±5.167 (n=5) inthe neutralizing antibody group, indication that the number ofosteoclast was significantly low in the neutralizing antibody group(P<0.05) (FIG. 24). With respect to the metastasis rate from primarytumor to lung, both groups showed metastasis, but the number ofmetastasized colonies was 89±28.9 in the control group and 30.5±6.30 inthe neutralizing antibody group, indicating that the number ofmetastasized colonies was significantly inhibited in the neutralizingantibody group (P<0.05) (FIG. 25). These results showed that theanti-rat Exon-17 polyclonal antibody not only inhibits primary tumorgrowth of breast cancer cells, but also has an inhibitory effect on lungmetastasis of breast cancer cells.

INDUSTRIAL APPLICABILITY

Diseases in which periostin is involved can be prevented and treated bysuppression of the function of a periostin isoform having anti-celladhesive activity highly expressed in a disease such as heart failure,inhibition of the aggravation of condition and improvement of thefunction of tissue by use of an antibody against the periostin isoformhaving anti-cell adhesive activity. Moreover, the presence of thediseases and the degree of progress of symptoms can be known bymeasurement of the amount of the periostin isoform in a sample from apatient. The use of antibodies against a peptide encoded by the Exon-17region of periostin enables cancer treatment, more specifically theinhibition of primary tumor growth and metastasis. It is also possibleto know the presence or absence of cancers and the degree of theirprogress by measuring the amounts of periostin isoforms in patientsamples using the above antibodies.

1. An isolated antibody against a periostin iso form having anti-celladhesive activity, specifically recognizing a site responsible foranti-cell adhesion of periostin, and capable of neutralizing anti-celladhesive activity of periostin.
 2. The antibody of claim 1, wherein thesite for responsible for anti-cell adhesion of periostin is the aminoacid sequence encoded by Exon-17 or by a part thereof.
 3. The antibodyof claim 2, wherein the amino acid sequence encoded by Exon-17 or by apart thereof is the amino acid sequence of SEQ ID NO: 3, 4, 21, 22, 23,24, 26, or
 34. 4. The antibody of claim 3, wherein the amino acidsequence is SEQ ID NO: 3, 4, or
 21. 5. The antibody of claim 1, whereinsaid antibody is a monoclonal antibody.
 6. The antibody of claim 5,wherein said antibody is produced by a hybridorna cell line FERMBP-10718.
 7. A hybridoma obtainable by a process comprising steps of:(a) immunizing a mammal with a peptide comprising the amino acidsequence of SEQ ID NO: 3, 4, 21, or a peptide introduced cysteineresidues into the N-terminus thereof; and (b) fusing anantibody-producing cell of the mammal with a myeloma cell.
 8. Ahybridoma cell line FERM BP-10718.
 9. A method for producing an antibodyof claim 5, comprising steps of: (a) immunizing a mammal with a peptidecomprising the amino acid sequence of SEQ IDs NO: 3, 4, and 21, or apeptide introduced cysteine residues into the N-terminus thereof; (b)fusing an antibody-producing cell of the mammal with a myeloma cell; and(c) culturing the obtained hybridoma.
 10. The method of claim 9, whereinthe hybridoma is a hybridoma cell line FERN BP-10718.
 11. Apharmaceutical composition comprising the antibody of claim
 1. 12.(canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)
 16. (canceled)17. (canceled)
 18. (canceled)
 19. A method for diagnosing a disease inwhich a periostin isoform having anti-cell adhesive activity isinvolved, comprising measuring the amount of the periostin isoform in abiological sample by using the antibody of claim
 1. 20. The method ofclaim 19, wherein the antibody is a labeled antibody.
 21. The method ofclaim 19, wherein the disease is heart failure, myocardial infarction,heart dilation, cardiac hypertrophy, cardiac fibrosis, cardiomyopathy,myocarditis, valvular disease, cancer, aneurysm, arteriosclerosis,central neurodegenerative disease, renal disease, rheumatoid arthritis,osteoporosis, pulmonary emphysema, pulmonary hypertension, chronicobstructive pulmonary disease (COPD), nephritis, pancreatitis,hepatitis, hepatic fibrosis, or pulmonary fibrosis.
 22. The method ofclaim 21, wherein the disease is cancer.
 23. (canceled)
 24. (canceled)25. (canceled)
 26. The method of claim 22, wherein the cancer ismalignant melanoma or breast cancer.
 27. A method for detecting orquantifying a periostin isoform having anti-cell adhesive activity in asample by using the antibody of claim
 1. 28. A diagnostic reagent for adisease in which a periostin isoforin having anti-cell adhesive activityis involved, comprising the antibody of claim
 1. 29. The diagnosticreagent of claim 28, wherein the disease is heart failure, myocardialinfarction, heart dilation, cardiac hypertrophy, cardiac fibrosis,cardiomyopathy, myocarditis, valvular disease, cancer, aneurysm,arteriosclerosis, central neurodegenerative disease, renal disease,rheumatoid arthritis, osteoporosis, pulmonary emphysema, pulmonaryhypertension, chronic obstructive pulmonary disease (COPD), nephritis,pancreatitis, hepatitis, hepatic fibrosis, or pulmonary fibrosis.